Fluconazole is a triazole antifungal agent used to combat secondary fungal infections such as cryptococcal meningitis, which persists in the membranes surrounding the brain and spinal cord.1 A lumbar puncture may only yield small volumes of cerebral spinal fluid (CSF) for performing diagnostics or determining exposure of fluconazole during treatment. Dried blood spot (DBS) or in this case dried matrix spot (DMS) analysis, was ideal for the small sample volume analysis (< 20 µL) of CSF. This DMS method, coupled with HPLC/MS/MS analysis, was developed for determining the fluconazole concentration in CSF samples.