Greg Halford was penalised for handball five minutes before the break and Keane's expertly taken spot-kick proved to be just about enough to overcome a determined Reading side, who we have now moved four points ahead of in the table.
Steve Coppell's side were not without chances themselves, Leroy Lita struck an upright with a header from close range, while Paul Robinson had a relatively active afternoon. Despite this, it was probably a game that should have been won by a more emphatic score,
With just a couple of minutes on the clock Reading were rocking slightly when Aaron Lennon played in Steed Malbranque to the left of the penalty area and the ball was drilled goalwards and skimmed the side netting. A better chance was to follow when Didier Zokora crossed and Dimitar Berbatov chested into the path of Keane in front of goal. The legs of Marcus Hahnemann denied what looked like being a likely opener.
The American was soon in action again when Malbranque picked out Berbatov inside the area and the goalkeeper quickly advanced to smother the intent of the Bulgarian. At the other end a Glen Little cross and header from five yards out from Leroy Lita probably should have seen us behind, but the England under-21 international struck the post.
Keane went for an ambitious chip when the creative Malbranque set him on the run again, Hahnemann was alive to the threat though, as was Robinson at the other end when the ball wasn't cleared with any great conviction and Dave Kitson was afforded a shooting chance from around penalty spot range.
It was a game of goalkeepers up to this point and Hahnemann made a smart save low to his left from Malbranque after Keane chested cleverly in his direction. Keane later had the ball nicked off his foot as he was about to strike, but he did have joy on 40 minutes.
Keane attempted to work himself some space from the close attention of Halford and the ball flicked up and struck the hand of of the former Colchester player. Ref Mr Wiley pointed to the spot and Keane fired his penalty high past Hahnemann.
Hahnemann did not emerge for the second half and was replaced in goal by Adam Federici. The pattern of the game remained much the same, the team continued to press but the final balls were not proving decisive enough and Reading did not suffer further deficit in the early part of the second period.
The ball did end up in the net on 58 minutes via Jermaine Jenas after Berbatov dinked back into the area following a Pascal Chimbonda cross, but the flag of the assistant was raised and JJ's joy short lived.
Reading then picked up a bit of momentum in their play and Kevin Doyle was added to the mix with 25 minutes left to make an impression. The next to do this was Keane when he latched onto a huge clearance from Robinson to go one on one with Federici for a high bouncing ball before Malbranque was not too far away with a drive from the edge of the area.
A second goal was proving elusive so it was deemed to be time for Defoe, who took over striking duties from Keane with 15 minutes left. The next chance fell to Berbatov though and Federici did well to pluck the ball away from the top corner of his goal.
Defoe wriggled free in the area but could not add enough bite to his shot to really trouble Federici before finding the net after clever play from Jenas but again the assistant's flag frustrated potential celebrations.
He should have netted in the closing minutes when Lee Young-Pyo teed him up inside the area, but his effort went high when he could have settled the game and prevented a final few nervous moments.
Spurs: Robinson, Chimbonda, Dawson, Rocha, YP Lee, Lennon, Jenas, Zokora, Malbranque (Huddlestone, 80), Berbatov, Keane (Defoe, 65). Subs: Cerny, Stalteri, Taarabt.
Reading: Hahnemann (Federici, 46), Halford, Shorey, Duberry, Ingimarsson, Sidwell, Little (Oster, 65), Harper, Lita, Hunt (Doyle, 65), Kitson. Subs: Gunnarsson, Bikey.
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A Robbie Keane penalty sealed victory over Reading at the Lane and made it five Premiership wins in a row.