This study aimed to assess the pain experienced during micropulse transscleral laser therapy (MPTLT) and overnight thereafter and explore the factors associated with the pain. This prospective study included 100 eyes of 81 glaucoma patients undergoing MPTLT under retrobulbar anesthesia. All patients were asked to rate both types of pain using a numerical rating scale (NRS). The risk factors were explored using multivariable mixed-effects ordinal logistic regression. The mean (SD) NRS pain score during the procedure was 3.57 (3.41) (range 0-10), which included no, mild, moderate, and severe pain in 30 (30%), 33 (33%), 17 (17%), and 20 (20%) eyes, respectively. The mean (SD) NRS score of overnight pain was 2.99 (2.28) (range 0-9), which included no, mild, moderate, and severe pain in 17 (17%), 59 (59%), 17 (17%), and 7 (7%) eyes, respectively. Twenty-seven (27%) eyes reported worse pain overnight than during the procedure. Increased age, initial intraocular pressure, and pain during the procedure were significantly associated with increased overnight pain (p < 0.05). Up to a fourth of eyes had worse pain after discharge. Older age, initial intraocular pressure, and pain during the procedure were risk factors for higher levels of overnight pain.