Background: Glaucoma represents a significant health problem and is an important cause of blindness worldwide. In order to delay the onset of advanced irreversible optic nerve injury and visual field abnormalities, topical medication treatment is typically used as first-line therapy. Objectives: To compare the effects of preservative-free and preservative-containing prostaglandin analogs on ocular surface by using impression cytology (IC) in newly diagnosed primary open angle glaucoma (POAG) patients. Methodology: A prospective randomized controlled clinical trial was done on 60 eyes of 30 patients with POAG and randomly divided into group A (n = 30) received preservative- free prostaglandin analogs and group B (n = 30) received preservative-containing prostaglandin analogs. Results: Clinical assessment of ocular surface features showed a significant difference between two study groups after 3 months of treatment. Schirmer test, tear film break up time (TBUT) and Fluorescein corneal staining (P<0.001). IC was significantly different between the two groups (P < 0.001). Also, the ocular surface disease index (OSDI) questionnaire score was significantly different between the two groups (P < 0.001). There was a significant difference in intra ocular pressure ( IOP) between the two study groups (P=0.018). Conclusion: Our study concluded that glaucoma patients treated with preservative-free prostaglandin analogs have less ocular surface side effects than those treated with preservative-containing prostaglandin analogs. This finding suggests improving the tolerability of the topical medications that reflected on the patient compliance, efficacy of treatment, and control of glaucoma.