The 25-year-old moved from Spurs in the transfer window but the deal was rushed through as a loan before he signed as a Pompey player early in January.
"The rules are clear," said a Premier League spokesman. "A player who is taken on loan and then during the course of the transfer window, if that loan is made permanent, he is not allowed to play against the team who originally loaned him."
Although the vote was unanimous, Spurs and Pompey did not appear to publicly acknowledge Defoe's absence until the eve of the clash at White Hart Lane on Saturday.
A Portsmouth FC spokesman said: "Clearly we are not happy about the situation and were under the impression it only applied to goalkeepers, following a similar situation last year with Tim Howard playing for Everton against Manchester United.
"Jermain was only signed on a loan deal due to time restraints on transfer deadline day and the deal was made permanent soon after.
"Spurs themselves were under the impression he was playing and clearly the rule needs to be looked at again as we will not be the only club to be affected by this.
"We have tried to get the ruling overturned and have lots of support from the other Premier League clubs but nothing can be done until the next shareholders' meeting in April."