The CTS-6 can be used clinically to diagnose carpal tunnel syndrome (CTS) and has demonstrated high levels of interrater reliability when administered by nonexpert clinicians. Our purpose was to assess sensitivity (Sn), specificity (Sp), and interrater reliability of the CTS-6 when administered by medical assistants (MAs). A series of patients presenting to an academic, upper-extremity surgery clinic were screened using CTS-6 between May and June of 2023. The CTS-6 was first administered by one of seven MAs and then by one of four fellowship-trained upper-extremity surgeons. In addition to recording baseline demographics, the results of each of the six history and examination components of the CTS-6 were recorded, as was the cumulative CTS-6 score (0-26). Surgeons were blinded to the scores obtained by the MAs. Interrater reliability (Cohen's kappa) was determined between the groups with respect to the diagnosis of CTS and the individual CTS-6 items. Sensitivity/specificity was calculated for the MA-administered CTS-6, using the score obtained by the surgeon as the reference standard. A CTS-6 score >12 was considered diagnostic of CTS. Two hundred eighteen patients were included, and 26% had a diagnosis of CTS. The MA group demonstrated a Sn/Sp of 84%/91% for the diagnosis of CTS. Interrater reliability was substantial (Cohen's kappa: 0.72, 95% confidence interval: 0.62-0.83) for MAs compared with hand surgeons for the diagnosis of CTS. For individual CTS-6 components, agreement was lowest for the assessment of two-point discrimination (fair) and highest for the assessment of subjective numbness (near perfect). The CTS-6 demonstrates substantial reliability and high Sn/Sp when administrated by MAs in an upper-extremity clinic. These data may be used to inform the development of CTS screening programs and future investigations in the primary care setting. Diagnostic II.