1) I hope you updated ALL statistics (index, column, etc) with a FULL SCAN immediately after upgrade. That is a MANDATORY step.

2) Most tuning will come exactly like it did in SQL 2000 - aggregate profiler runs.

3) It is MUCH easier to do wait stats and file IO stall analyses now, and both of those should be a primary tool to find where it hurts on a wider scale.

4) sp_whoisactive: get it, learn all of it's goodness. it is documented inside the code and Adam Machanic did a 30-day blog post series on sqlblog.com

5) LOTS of Dynamic Management Views to help you. There are some good books about using them for tuning. Performance Tuning with SQL Server Dynamic Management Views for example, and Troubleshooting SQL Server - A Guide for the Accidental DBA. There are numerous more advanced books too, as well as a bunch of free eBooks.

6) Consider getting a performance tuning professional on board to give your systems and applications a review. Probably lots of low-hanging fruit can be found and fixed plus you would gain the invaluable benefit of being mentored by a guru on how to do effective tuning analysis and refactoring.