The South Africa international has been struggling with a groin problem that first came to light last month.
Pienaar represented his country during the recent international break, but he revealed that the injury had flared up again, writing on his Twitter account: "Out for the Madrid game. Groin still not right."
If confirmed by the club's doctors, the news will come as another blow for Spurs boss Harry Redknapp, who is already sweating on the fitness of defender William Gallas and star winger Gareth Bale.
Ledley King, Jonathan Woodgate and Younes Kaboul are all definitely out of the match, while midfielder Tom Huddlestone is only just back from a long-term lay-off.
Pienaar was named among the substitutes for Saturday's goalless draw with Wigan at the DW Stadium but did not get on the pitch.
Source: PA
Source: PA