Khan has already had to defend the appointment after Assembly Members raised questions over controversial comments made by Lame on Twitter.
The mayor stressed on Wednesday that he “would not endorse of condone” comments by Lame that included references to "Tory scum", expressing disappointment that the Queen is still alive, and fantasising about "bitch-slapping" David Cameron and Baroness Warsi.
And now Assembly Member Shaun Bailey is demanding the new Night Tsar attend a scrutiny committee to answer questions over her plans for the role.
Writing to economy committee chair Fiona Twycross, Bailey said: “The role of Night Tsar has the potential to be transformational for London, and therefore it is fundamentally important that the first person to hold that role has the chance to explain to the London Assembly her 'plans' at the earliest opportunity.
"This will also give Ms Lame a chance to address members' concerns and prove to us that she will be capable of representing all Londoners."
Twycross, a Labour AM who also serves as chair of the London Fire and Emergency Planning Authority under Khan, will have to approve the decision to call Lame.
The London Assembly's oversight committee, chaired by London Tories leader Gareth Bacon, is already hosting a hearing looking into the recruitment of Lame next week.
Separately, Kingston and Surbiton MP James Berry has also written to Khan to ask him to justify the decision to pay the Night Tsar's £35,000 salary through a limited company, arguing this is a regualrly used tactic to minimise contributions to HMRC.
In a letter Berry said: “You agreed in Wednesday’s Question Time, with Dame Margaret Hodge’s statement that “avoiding tax when paying public sector staff is almost always staggeringly inappropriate”, so I ask that you stand by that statement and reverse your decision to pay Amy Lamé through a personal service company.”