BACKGROUND The standard approach for benzodiazepine detection often includes immunoassay followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS). The illicit use of non-prescribed benzodiazepines has been trending up nationally. METHODS We developed and validated an improved LC-MS/MS assay for benzodiazepine detection in urine. We expanded the testing panel by adding five drugs to the previous panel of ten. We determined the prevalence of individual benzodiazepines in our patient population. Immunoassay results were compared with LC-MS/MS to evaluate assay performance. RESULTS Clonazepam and alprazolam were the most common benzodiazepines present. Etizolam and flualprazolam were also prevalent in Washington State. Compared with the LC-MS/MS assay, the immunoassay had variable cross-reactivity, which explained false negative and false positive immunoassay results. The inclusion of new drugs in the LC-MS/MS panel significantly reduced the incidence of immunoassay results interpreted as falsely positive. CONCLUSIONS New illicit benzodiazepines have emerged regionally and nationally. The inclusion of novel drugs in LC-MS/MS assay was helpful in properly characterizing the epidemiology of benzodiazepine use in our patient population. This information will lead to better assay result interpretations and patient care, and our experiences provide a roadmap for other clinical laboratories looking to expand their testing menu or transition to new instrumentation.