AIM: To validate the Peek Acuity mobile phone application in pediatric populations and compare its utility, both economic and diagnostic, against conventional screening methods using a pediatric ophthalmologist examination as the gold standard. METHODS: A cohort of 393 subjects from Fernando de la Mora, Paraguay (ages 6-16y) were enrolled in the study. Subjects were randomly assigned a starting screening modality among: Peek Acuity, a single line of tumbling E optotypes set at 20/40, and Spot Vision Screener. Once completing the first screening modality, the subjects completed the two remaining techniques. Referral criteria were established based on the most current American Association of Pediatric Ophthalmology and Strabismus (AAPOS) recommendations: 20/40 for Peek Acuity and the tumbling E, and refractive error detection for the Spot Vision Screener. Subjects that failed to achieve the cut-off for any of the three screening techniques or subjects that passed the screening but were randomly selected to perform a comprehensive eye exam to determine the false negative rate, were evaluated by a pediatric ophthalmologist. This evaluation was considered the gold standard, and included vision assessment by a Snellen chart, strabismus evaluation, and cycloplegic refraction with dilated fundoscopy. RESULTS: We obtained 48% sensitivity, 83% specificity, 43% positive predictive value, and 86% negative predictive value for Peek Acuity's ability to refer compared to evaluation by a pediatric ophthalmologist, failing to achieve a desired sensitivity for implementation. Peek Acuity trended to overestimate the subject's visual acuity, providing a higher visual acuity that would not indicate referral for a comprehensive eye examination. However, its high specificity accurately predicted a significant number of children who did not need further evaluation. When comparing the three screening methods, no single screening modality outperformed the others. Peek Acuity represented a technology that was economically feasible compared to other screening modalities in low income settings, due to the prevalence of cell phone use. CONCLUSION: Peek Acuity represents an efficient tool that has potential for implementation in school screenings with different strategies aimed at pediatric populations due to its low cost and high specificity. An increase in sensitivity would improve detection of children with refractive errors.