Background: 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. Methods: 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. Results: 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). Conclusions: 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.