2nd Trip to Italy here is our Itinerary I am open to ideas /suggestions

Is your cooking class already booked? I don't know how jet lag affects you, but I think I'd be a bit too incoherent my first day for a cooking class - you might want to book it a bit later :)

You may consider Perugia, not far from Assi. It is a lovely city with a great art musem. It is worth a night there. It is up on a hill, but luckily there's a tram from the train station that takes you to the top.
As for Tuscany, we used some of the routes recommended by the DK Eyewitness Guide Backroads Italy book. They talk about the most scenic roads and places to see along the way. I was able to get it from my library. A restaurant recommendation is Ristoro di Lamole, in the tiny village of Lamole in the Chianti region. Great food and a lovely terrace. Reservations recommended.

my husband wants to see where Paul was imprisoned

I last visited the Mamertine prison site about 15 years ago. It was sparse but dramatic and moving. But since then, according to Rick's Italy guidebook, the site has been taken over by a commercial bus company and turned into a multi-media tourist trap, literally not worth the price of admission. Do some research and decide for yourself, but you might be better off staying outside and exploring the Imperial Forums.

After that heading to B&B in Assisi. Day 4: Winery tour that's it. Day 5 and 6 nothing planned so far except visiting Basilica maybe?

Assisi is wonderful. The magic is at night, especially. Explore the streets, marvel at the floodlit Basilica, and gaze out at the lights of Santa Maria degli Angeli in the valley below. There are several excellent restaurants; our favorites are La Pallotta, just off Piazza del Comune; and Trattoria da Erminio. When the day-trippers crowd the area near the Basilica, bug out and hike up to Rocca Maggiore, or take the bus down to Santa Maria degli Angeli.

Try the Tuscan Countryside it deserves a good 5 days South of Firenza you have Siena and te Beautiful Val d'Orcia, Montepulciano & Montalcino where te wine is simply spectacular.
If you find yourself to be around Montalcino please make sure you visit the CAPANNA WINERY. It as been amongst the first 7 Brunello producers in the region, you'll will be trilled by the autenticity of te experience.

If you have never been to Rome before, it seems you don't have ample time there compared with other locations.
You only have 2 nights in Rome I believe and as mentioned the first day you will jet lagged so have your 1 full day starting early with the Vatican, ending with the Collesseum in what surely will be a tiring day.
Another night in Rome I thought would be quite beneficial.

I think your second day 8 and 9 should be days 10 and 11?
The summarized plan 2 nights Rome, 3 nights Assisi and 5 nights in Tuscany is that accurate? (though it is a big area and you did not name a town). Depending on the town location Day 9 could be far or Day 10 could be far.
Siena could be an option for your open Day 7

Spello and Trevi are beautiful hill towns a couple of train stops from Assisi, but a car will be more convenient.

In Tuscany, Arezzo, Siena, Montalcino - all beautiful.

Susanne, I'd agree with lnbsig that scheduling a cooking class on arrival day is risky...not only because you may be too cross-eyed with jet lag to pay attention but because it's never certain that flights are going to arrive when they're supposed to. Up to you, of course, but I'd opt for some other activities - outside, preferably - to stay awake.

The Mamertine: throwing my vote in with Jeff to skip this. It's been closed for awhile and is re-opening this week but the previous version was a waste of time and money, and (sorry but you're talking to a skeptic here) there's no historical proof that Biblical prisoners were ever held here. We did go strictly for the archeological value and found little to recommend: much better things to do, IMHO. Granted, we saw the prior (terrible) presentation so I've no idea what they're doing with the updated version but I see that they're more careful with their 'historical' claims than they were, and that it has seen some additional excavation. "Reconstruction of the original environment" gives me pause, though. It will only be open 4 days a week:

http://www.operaromanapellegrinaggi.org/en/roma-cristiana/carcer-tullianum

Doing the Vatican and the Borghese in one day would be exhausting but if you love art and museums, the Borghese is a MUST. We enjoyed that one much, much more than the Vatican for excellent crowd control, quality/accessibility of the collection and fascinating building. It only take two hours, and I'd try really hard to get that in somewhere.

There are any number of outstanding churches with fabulous art in Rome as well. Only two days for all that the city has to offer is cutting it really short if your Other Half has never been there?

And again for art, it's a shame to only give Florence a single day? As the cradle of the Italian Renaissance, it's simply stuffed with glorious art. To be honest, the Accademia's 'David' was, for me, the least of it (Bernini's in the Borghese is a marvel.) I assume the Uffizi is included in your WOI tour?

Thanks guys for all the great tips! As for the cooking class, its very informal at our B&B where we are staying and it will be our first dinner in Italy. Our flight leaves at 11 pm and we will sleep on the flight to Italy. I have traveled many times overseas and yes its tiring but manageable. The town of Florence needs days not like we have planned 1/2 day. But my husband only tolerates looking at stuff for so long and this is also a romantic trip so we need time for picnics and so on. I booked our last 2 B&B with pools as we like to just hang out , sip wine and enjoy the countryside. On our next trip I plan on renting a house instead and revisit Florence for longer.