Purpose: To analyze the ocular surface changes in eyes after the withdrawal of anti-glaucomatous drugs when non-penetrating deep sclerectomy (NPDS) is performed. Methods: Thirty-one patients (33 eyes) diagnosed with glaucoma that underwent NPDS were included in this prospective study. The control group included 33 eyes. Four variables were studied using Keratograph 5M (K5M): ocular hyperemia (OH), non-invasive tear film break-up time (NI-BUT), lower tear meniscus height (LTMH), and meibography. LTMH was also measured using the anterior segment module of a Spectralis Fourier-domain optical coherence tomography (FD-OCT) instrument. Moreover, an evaluation of corneal and conjunctival staining was performed. In the glaucoma group, five visits were carried out: pre-surgery, 1 week after surgery, and 1 month, 3 months, and 6 months after surgery. In control groups, examinations were performed in only one visit. In addition, patients were asked to answer two questionnaires: Ocular Surface Disease Index (OSDI) and National Eye Institute Visual Functioning Questionnaire (NEI VFQ-25) before and 6 months after surgery. Results: Before NPDS, eyes showed worse objective data than healthy control subjects (P <= 0.049). In this group, a significant improvement was observed in questionnaire responses (P < 0.001), LTMH-FD-OCT (P = 0.037), LTMH-K5M (P = 0.025), K5M-OH (P = 0.003), NI-BUT (P = 0.022), and conjunctival and corneal staining (P < 0.001). No significant differences were observed between groups in FD-OCT and K5M LTMH, NI-BUT, corneal-conjunctival staining, nor in the most OH sector values at 6 months (P & GE; 0.62). Conclusion: A significant improvement in the ocular surface was observed 6 months after NPDS, suggesting that the withdrawal of the topical anti-glaucomatous treatment had a beneficial effect on the subjects.