Just as in the opening game against Paris Saint-Germain, the Giallorossi came from behind to secure a draw with their opponents - only to lose the ensuing penalty shootout.
Edin Dzeko scored Roma's goal midway through the second half, as he latched onto Aleksandar Kolarov's well-weighted cross to divert home. Kevin Strootman also crashed a shot off the bar but, after Mario Mandzukic's early strike, the game ended in a draw.
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Coach Eusebio Di Francesco's side got off to a bad start in the shootout, as substitute Marco Tumminello - the last-minute hero in the win over Spurs - saw his attempt saved.
Lorenzo Pellegrini, Juan Iturbe, Bruno Peres and Cengiz Under all subsequently converted theirs but, with Juventus proving perfect from 12 yards, those efforts would prove to be in vain.
Nevertheless, having emerged undefeated over 90 minutes from the three games in the United States, Di Francesco will be content with the performances shown by his side.
The squad will now return to Rome, before travelling to Spain next week ahead of further pre-season encounters against Sevilla and Celta Vigo.
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JUVENTUS WIN 5-4 ON PENALTIES
Goalscorers: Dzeko (74'); Mandzukic (29')
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