Pettitte struck out two in the Florida State League game against Clearwater. He threw 26 of 32 pitches for strikes.
"I felt good with everything," Pettitte said. "Just another good step."
Pettitte said the current plan for him is to make another start with Tampa on Sunday.
Injured Phillies reliever Jose Contreras, coming back from elbow surgery last September, also pitched in the game. The right-hander struck out two during a 1-2-3 inning.
Pettitte worked a scoreless inning in the Yankees' spring training finale last Wednesday against the New York Mets. The left-hander agreed to a minor league contract on March 16 and could be ready to join the Yankees in May.
"This is a process," Pettitte said. "It's going to take a little bit of time. My legs felt really good under me. We'll bump it, probably to 50 pitches hopefully next time, and we'll see what happens from there."
After needing just eight pitches in a perfect first, Pettitte gave up a second-inning lead-off double to Brandon Tripp, who advanced to third on a wild pitch and scored when Jeremy Barnes hit a sacrifice fly.
"It's good to get out there and get in the stretch, and have some of those guys on," Pettitte said. "Working on the slide-step and just getting in a rhythm with that. I haven't had a chance to really work on all my pitches from the slide-step and from my regular leg lift when runners are on base."
Clearwater's Miguel Alvarez opened the third with a double that took a high bounce into the outfield after hitting the third-base bag. Pettitte worked out of the jam by retiring the next three batters.
"This was a short sample, a short outing, and feel like maybe I didn't get a chance to throw some of the pitches that I would have liked to throw because I'm getting some guys out in early counts," Pettitte said. "But all-in-all I feel real comfortable with everything."
Yankees reliever David Aardsma, coming back from elbow-ligament replacement surgery, made 30 pitches during his second throwing session on a 5-inch, half-mound. ... New York RHP Adam Miller, who made just 31 relief appearances over the previous three seasons because of an injured right middle finger, threw two innings in an extended spring training game. ... RHP Brad Meyers, an offseason Rule 5 pick on the disabled list due to a strained right shoulder, followed Pettitte and gave up eight runs and eight hits in 1 2/3 innings.