The chant could be heard in the second half of the Carabao Cup semi-final first leg at Stamford Bridge.
In August, anti-discrimination group Kick It Out condemned the same chant by Liverpool fans aimed at Norwich's on-loan Chelsea midfielder Billy Gilmour.
A Spurs statement this morning read: "The club is extremely disappointed by homophobic chanting from sections of our support at Stamford Bridge on Wednesday night.
"We work closely with our LGBTQ+ fan group Proud Lilywhites to create a welcoming and inclusive environment at our Club and are proud to display the Progress Flag in our stadium on matchdays.
"No one should suffer discrimination because of their sexual orientation and / or their gender identity and we urge supporters not to use this chant.
"Homophobic chanting at Tottenham Hotspur Stadium should be reported by texting HPH and location details to 07537 404821. At other stadia it should be reported to the nearest steward or by the home club’s reporting number.
"There is no place for discrimination at Tottenham Hotspur."
Liverpool described the chants aimed at Gilmour as "offensive and inappropriate", while a Kick It Out statement at the time read: "The use of the term 'rent boy' in chants by a portion of Liverpool fans at Carrow Road was unquestionably homophobic and has no place in our game. We have also had reports of incidents at other games and are awaiting further clarification."
