The Italian giants came to White Hart Lane on the back of a five-match winning streak, but they were outplayed for large parts of Thursday night's Group J clash, which Villas-Boas clearly feels his side should have won.
The Portuguese, who earned his first win as Spurs boss on Sunday, was livid at referee Ovidiu Alin Hategan for disallowing Steven Caulker's goal deep into the second half.
The centre-back leapt high to head home Gareth Bale's corner but the Romanian official ruled he had pushed his marker in the lead-up to the goal, a ruling which caused Villas-Boas to fling his arms up in the air in an angry manner.
The Portuguese had already seen Clint Dempsey's header ruled out for a marginal offside and Bale also had a strike chalked off after latching on to the impressive American's through-ball.
Sandro and Bale both missed close-range headers and Villas-Boas will probably look back at this as a missed opportunity ahead of what is expected to be a tough trip to Panathinaikos next month.
Lazio could have stolen the game at the end through Stefano Mauri and came within a whisker of going ahead in the first half when Alvaro Gonzalez beat Hugo Lloris but saw his shot rattle the woodwork.
Aaron Lennon, captain for the night, Gareth Bale and Caulker all impressed while a clean sheet for debutant Lloris is another plus point.
All eyes were on the Frenchman early on as he was chosen ahead of Brad Friedel for the encounter. The £8million signing seemed nervous early on when he bowled the ball out to a shocked Kyle Naughton, but the full-back was fouled by an over-exuberant Ogenyi Onazi.
Lennon latched on to a defence-splitting pass from Kyle Walker in the first chance of the match, but his cross was cut out by Luis Cavanda as Bale lurked at the back post.
Dempsey claimed handball when his shot was blocked by Giuseppe Biava on the edge of the box but Hategan waved play on.
A Walker mistake allowed Gonzalez to break free down the left but his cross evaded the advancing Miroslav Klose.
Tottenham's best chance of the first half came after 18 minutes when Jermain Defoe clipped a lofted ball to the back post where Bale was waiting - but the Welshman headed straight at Federico Marchetti.
Bale thought he had made amends for his miss moments later when he delivered a pinpoint cross which Dempsey converted with a diving header, but the linesman flagged for a very tight offside call.
Lazio started to come back in to the game however. Mauri knocked Cristian Ledesma's pass in to the path of Klose, but the German completely missed the ball, then four minutes before the break Gonzalez volleyed Jan Vertonghen's clearance against the bar with Lloris beaten.
Tottenham came out much more confidently after the break and they nearly took the lead seven minutes in when Defoe played in Lennon. Marchetti parried the winger's shot into Dempsey's path but he made a hash of the follow-up.
Spurs had the ball in the net for a second time just before the hour but again the goal was ruled out after the linesman ruled Bale had drifted offside when he latched on to Dempsey's through-ball.
Lazio had no answer to the pace of Bale and Lennon, who were dominating the flanks.
Andre Dias was booked for hacking down Bale after 61 minutes and Gonzalez saw yellow a few minutes later when he took Lennon out after the winger had given him the slip.
Sandro should have put Spurs in front after 64 minutes when he was picked out by Bale but the unmarked Brazilian headed wide from six yards.
Dempsey thought he should have had a penalty soon after when he appeared to be pushed in the box but again referee Hategan ruled against the hosts.
The official was in the spotlight again moments later when Caulker bundled in Bale's corner but he decided the defender had climbed on the back of his marker - prompting Villas-Boas' outburst.
Mauri beat the offside trap to slip into the Tottenham box in a rare Lazio attack but Caulker scampered back and denied the midfielder.
Mauri almost won it for Lazio five minutes from time when he sneaked through the Spurs defence but he could not finish.