Defoe, 31, did not train ahead of the game because of a hamstring injury and Spurs head coach Tim Sherwood confirmed he would not be ready in time to face the Ukraine outfit.
The England forward travels out to Toronto to begin his Major League Soccer career at the weekend, denying him the chance to bid farewell to Spurs with one final appearance.
But Sherwood has been boosted by the news that Brazil midfielder Sandro could return for the European fixture.
Spurs will attempt to overturn the 1-0 deficit from Ukraine when Sandro was again unavailable with the calf injury which has kept him out of Tottenham's last 14 matches in all competitions.
The Brazilian picked up the problem against Liverpool on December 15 but played 60 minutes in the Spurs Under-21s' last outing against Bolton before taking a full part in training this week.
"Sandro is looking good," said Sherwood.
"We have to be cautious with him because he's broken down before but he's done more training than he's done previously and we believe he's ready."