Purpose To evaluate the tear film osmolarity (TFO) and ocular surface clinical signs and symptoms in chronically medicated glaucoma patients and post-trabeculectomy patients. Methods This is a single-center, prospective case-controlled study. One-hundred and thirty eyes of 130 participants aged Z45 years were included (49 normal controls, 50 glaucoma patients on chronic preserved antiglaucoma medication Z6 months, and 31 post-trabeculectomy patients not on medication Z6 months). TFO, tear break-up time (TBUT), Schirmer's test I and dry eye symptoms were evaluated. Data from both groups of glaucoma patients were compared with age and sex-matched controls. Logistic regression was performed to calculate the odds ratios. Results Mean TFO in the three groups were 301.4 ± 7.7, 307.0 ± 9.3, and 307.4 ± 11.6 mOsm/l, respectively. Compared with normal controls, chronically medicated glaucoma patients and post-trabeculectomy patients were more likely to have a raised TFO, with odds ratios (95% CI) of 4.43 (1.74-11.32) and 2.76 (1.02-7.94), respectively. Both groups of glaucoma patients were also more likely to experience dry eye symptoms, with ORs of 4.72 (1.92-11.59) and 4.24 (1.54-11.72). There was no significant difference in TFO and symptoms between both groups of glaucoma patients, and in TBUT and Schirmer's test across all three groups. Conclusions Patients on chronic topical antiglaucoma medication and posttrabeculectomy patients were more likely to have raised TFO and dry eye symptoms, suggesting significant ocular surface disease. Glaucoma practitioners should be aware that dry eye symptoms and raised TFO may occur in the absence of TBUT and Schirmer's test abnormality.