The previous meeting between the sides had seen the Hatters ease through 4-0 in an FA Cup tie, but they had to work much harder at Nethermoor Park against the Blue Square Bet North high-flyers, who will feel they should have at least earned a replay.
Guiseley came into the tie on the back of their best FA Trophy run for 17 years.
Having already disposed of holders Barrow and Eastbourne Borough in this season's competition, they were confident of causing another upset against the Hatters and returning to the semi-finals.
That previous best had seen the likes of then-Conference teams Cheltenham Town and Morecambe fall by the wayside before the Yorkshire outfit went down to Runcorn over two legs. Keen to avoid a similar giant-killing, Luton boss Richard Money named a side that was still strong despite six changes.
Nevertheless, the hosts took the game to their more illustrious opponents and came close to taking the lead in the third minute when Warren Peyton curled a shot just over the bar from 18 yards.
Luton were dealt a blow nine minutes later when Alex Lawless hobbled off with an injury. He was replaced by Claude Gnakpa, with Amari Morgan-Smith moving into central midfield.
However, they perhaps should have made more of an opening on 24 minutes.
Barnes-Homer broke down the left wing, beating the offside trap to latch onto a clearance, but his low cross aimed for Jason Walker was cleared by Simon Ainge.
Guiseley countered and would have surely broken the deadlock had Joe O'Neill been able to get a touch on a strike from just outside the box from James Walshaw.
Both sides were enjoying plenty of possession, although the Hatters were struggling to carve out clear cut chances.
Jake Howells did manage to pick out substitute Gnakpa with a cross, but he shot wide under pressure from Dave Merris on the half hour.
The best chance of the half for the visitors fell Morgan-Smith. He went on a barnstorming run from the halfway line before unleashing a shot from 25 yards that Guiseley goalkeeper Steven Drench parried away. Gnakpa went in for the follow-up, but Drench saved again.
As the first 45 minutes drew to a close, the home side were awarded a free kick 25 yards out when Keith Keane was deemed to have fouled O'Neill. The striker got up to take the kick, but shot straight at Kevin Pilkington. The Hatters goalkeeper also denied Walshaw as the sides entered half time all square.
Manager Money opted to bring on Danny Crow for Walker at half time and the replacement made an immediate impact, providing a perfectly-weighted through ball for Barnes-Homer to latch onto before placing the ball into Drench's bottom right hand corner on 49 minutes.
Guiseley were momentarily shaken by this turn of events, having started the second period brightly and feeling they were denied a penalty when Walshaw and Zdenek Kroca came together in the box.
Having regained their composure, the hosts went back on the attack and won a free kick from a similar distance as their earlier one. This time it was Peyton who took it and saw his attempt just whistle past goalkeeper Pilkington's left post.
Meanwhile, the Hatters were looking to put the game to bed. Drench had a routine save to make from a Kroca header on 67 minutes, but two minutes later did well to get down and save a shot from Howells after good work from Crow and Barnes- Homer.
Guiseley's persistance was reaping small rewards though and in the 77th minute Pilkington did well to tip a fiercly-struck Peyton free kick over the bar with a onehanded save.
Determined to secure at least a replay, home boss Steve Kittrick brought on forwards Darryn Stamp and Luke Sharry in a double substitution.
Guiseley also had a second penalty shout turned down by referee Stephen Martin in the closing minutes when Walshaw took a tumble in the box.
But Luton held on for a narrow win and it is them who go into the hat for Monday's semi-final draw.
Luton Town: K. Pilkington; Graham, G.
Pilkington (captain), Kroca, Murray; Morgan-Smith (Poku 60), Lawless (Gnakpa 12), Keane, Howells; Walker (Crow 46), Barnes-Homer Used subs: Tyler, O'Donnell Referee: Mr. Stephen Martin (Staffordshire)