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Purpose: To validate the Spot Vision Screener, a handheld digital screening device that evaluates children for amblyopia risk factors as defined by 2013 criteria of the American Association for Pediatric Ophthalmology and Strabismus (AAPOS), in the setting of a controlled pediatric ophthalmology clinic., Methods: During a 3-month period, children 2-9 years of age were screened using Spot in a pediatric ophthalmology clinic before receiving a gold standard eye examination. Gold standard examinations were evaluated using the 2013 AAPOS Vision Screening Committee guidelines and compared with results from Spot, which were evaluated using two different manufacturer referral criteria: v1.0.3 and v1.1.51. The specificity and sensitivity for each set of referral criteria to detect both amblyopia risk factors and amblyopia were calculated., Results: A total of 233 children were included. Of these, 155 were successfully screened and analyzed according to two different referral criteria. Spot screeing revealed ambyopia risk factors in 109 patients; examination confirmed amblyopia in 64. Using the original manufacturer's criteria (v1.0.3), Spot was 89% sensitive and 71% specific in detecting amblyopia risk factors. The updated referral criteria (v1.1.51) were applied to the same 155 patients, and specificity improved to 88% (P < 0.02); sensitivity remained minimally affected, at 85% (P < 0.05). Spot-v1.0.3 was 92% sensitive and 41% specific in detecting amblyopia, whereas Spot-v1.1.51 was 89% sensitive and 53% specific for detecting amblyopia., Conclusions: The Spot-v1.0.3 had high sensitivity but overreferred for suspected myopia and strabismus; Spot-v1.1.51 maintained high sensitivity and improved specificity. The original referral criteria has a high sensitivity to detect amblyopia risk factors but low specificty; v1.1.51 criteria increases specificity with minimal impact on sensitivity., (Copyright © 2014 American Association for Pediatric Ophthalmology and Strabismus. Published by Elsevier Inc. All rights reserved.)