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The laboratory diagnostic detection of herpes simplex virus (HSV) from eye samples must be practical, timely, and definitive for appropriate therapy. Although polymerase chain reaction (PCR) and/or cell culture can be definitive, HSV results can be delayed. Enzyme Linked Virus Inducible System (ELVIS) is a test that can provide results within 24 to 48 hr. We evaluated “AmpliVue HSV 1+2 Assay” as a molecular colorimetric test that can detect HSV (1 or 2) DNA within 1 hr.Cornea/conjunctival samples were tested retrospectively with AmpliVue against 53 true-positive and 20 true-negative specimens collected in chlamydial transport medium. All clinical specimens were tested by cell culture isolation, PCR, and ELVIS for routine patient care.The sensitivity of AmpliVue against ocular samples that were both culture-positive and PCR-positive was 84%. The specificity of AmpliVue was 100%. Only one clinical sample was HSV-2 positive, whereas all others tested positive for HSV-1. Based on PCR-positive and cell culture-negative samples, AmpliVue (11 of 17) tested more positive than ELVIS (0 of 17) (P=0.003, Fisher Exact).AmpliVue is moderately sensitive and highly specific as a practical and timely diagnostic test for detecting ocular HSV. Expertise is readily achieved and the test is straightforward with easy interpretation. Negative AmpliVue testing must be confirmed with PCR. AmpliVue has potential as an office-based diagnostic test.