Barça ran riot in the first-half, cruising to a two-goal lead.
However, an inspired Spurs comeback lead by Christian Eriksen took the game all the way to penalties.
Spurs youngster Anthony Georgiou's penalty miss proved the decisive spot-kick as Barcelona new boy Malcom converted to secure victory for the Blaugrana.
Tale of two halves
For the second game in a row, Spurs conceded early on.
A well-worked move from a throw-in was headered home by Munir El Haddadi at the far post.
The high intensity shown by the La Liga champions paid off as they found Tottenham's defence sleeping for the opening goal.
Argentine goalkeeper, Paulo Gazzaniga denied Barça from doubling their lead before the half-hour mark after a fierce strike from Nélson Semedo was parried wide.
It took just a matter of minutes later for Arthur Melo to put Barcelona two goals ahead. On his Barça debut, the youngster scored the goal of the tournament after cannoning home from 25 yards out.
The opening 45 minutes provided a stark contrast to the performance Spurs provided in Wednesday's 4-1 victory over Roma.
Slow, laboured and unimaginative, Tottenham were deservedly behind with their only shot on goal coming from a Christian Eriksen free-kick.
The Londoners next effort on goal came in the early stages of the second half when Eriksen's free-kick cannoned off the post to safety.
Spurs had a new lease of life about them in the second period, on top for the majority of the half the Londoners enjoyed the lions share of possession.
Heung-min Son halved the deficit with just over 15 minutes to play after slotting home a rebound from an Eriksen shot. The Dane was denied by Jasper Cillessen but the South Korean made no mistake in converting.
Almost straight from the kick-off, Spurs drew level thanks to Georges-Kévin N'Koudou. Another scrappy goal for Spurs but thoroughly deserved based on their second-half performance as N'Koudou volleyed the ball home from close distance.
But Spurs could not find a winning goal in regulation time despite their second-half dominance meaning a penalty shoot-out would decide the tie.
Will Spurs be made to pay?
Despite an improved second-half performance from Mauricio Pochettino's men the Lilywhites, the lack of options were evident.
Tottenham's greater fitness became evident in the second period of the game with Barça seeming to run out of steam.
Although the result will not be heavily looked into, Tottenham's lack of options from the bench indicates their need to strengthen in this transfer window.
In particular, Spurs' central midfield was the area in which was overran in the game. with Mousa Dembele and Eric Dier still on a break and Victor Wanyama injured it is certainly an area in which Spurs must improve.
Tottenham now face their last International Champions Cup game against AC Milan on Wednesday 01:35 am (BST).