Nole failed in attempt to win his third title in New York, and his 13th career GS crown, when he lost to no.3 seed Stan Wawrinka 7-6 (1), 4-6, 5-7, 3-6.
Novak continued his pursuit of a 13th Grand Slam title with a 6-3, 6-2, 3-6, 6-2 win over no.10 seed Gael Monfils.
World no.1 moved one step closer to his 13th Grand Slam on Tuesday after Jo-Wilfried Tsonga retired in the third set (6-3, 6-2).
Novak booked his spot in the last 8 for the 10th time after dismissing Kyle Edmund 6-2, 6-1, 6-4 on Sunday night at Flushing Meadows.
The world no.1 got pass into the US Open fourth round as Mikhail Youzhny retired injured at 4-2 for the defending champion.
The top seed and defending champion will face Mikhail Youzhny (RUS) in the third round on Friday.
Nole began his quest for a third US Open title with a 6-3, 5-7, 6-2, 6-1 victory over Jerzy Janowicz at Flushing Meadows on Monday night.
Top seed and defending champion will open campaign in his 12th US Open appearance against Jerzy Janowicz (Poland).
The 2016 US Open draw will be led by reigning champion Novak, followed by Olympic gold medalist Murray, Wawrinka and Nadal.
Novak has withdrawn from the Western & Southern Open due to recurring injury. His next event will be the US Open.