Objective: To design and validate a questionnaire on the knowledge of primary open-angle glaucoma (POAG) in Spanish-speaking patients. Methods: An observational study was conducted on adults with diagnosis of POAG and who received outpatient medical care in a national reference health care institute of Peru. The questionnaire was based on a literature review and expert judgment. A pilot study was carried out on 60 patients. The items were classified and weighted according to the expert judgment. The final questionnaire was completed by 314 patients and its overall score was scaled by tertiles. Results: A total of 72 items were found by primary searching. The initial questionnaire was made up of 20 items, which was tested in a pilot study. A total of 14 items were selected that were then classified into 3 weighted domains: risk factors (10%), clinical aspects (36%), and treatment (54%). The questionnaire has a score range from 0 to 100 points (lowest to highest level). In patients, the mean score was 62.6 ± 14.1. The overall score was classified into 3 categories of knowledge level: low (0 to 55 points), medium (56 to 69 points), and high (70 to 100 points). Conclusions: The questionnaire provides evidence of validity to assess knowledge of POAG in Peruvian Spanish-speaking patients.