Diagnostic testing for Zika virus infection can be accomplished using molecular and serologic methods. Real-time reverse transcription-polymerase chain reaction (rRT-PCR) is the preferred test for Zika virus infection because it can be performed rapidly and is highly specific (1,2). However, in most patients, Zika virus RNA is unlikely to be detected in serum after the first week of illness (2,3). Recent reports using adaptations of previously published methods (2,4) suggest that Zika virus RNA can be detected in urine for at least 2 weeks after onset of symptoms (3,5-7). Currently, the CDC Trioplex rRT-PCR assay is the only diagnostic tool authorized by the Food and Drug Administration for Zika virus testing of urine (1). Other laboratory-developed tests will need in-house validations to adequately characterize the performance of the assay and meet Clinical Laboratory Improvement Amendments requirements. Further investigation is needed to determine the sensitivity and utility of Zika virus rRT-PCR on urine specimens collected ≥14 days after onset of symptoms.