Objective To establish a sensitive, rapid and simple high-performance liquid chromatography (HPLC) method for the determination of amphotericin B (AraB) and itraconazole (ITZ) in human cerebrospinal fluid (CSF). Methods Solid-phase extraction (SPE) was involved in sample preparation in this method, followed by reverse-phase separation under two different chromatographic systems. Results The tinearity for both AraB and ITZ ranged from 5 ng/mL to 100 ng/mL and the inter-day and intra-day RSD were both below 7.6% at the concentrations of 20,50 and 100 ng/mL. Conclusions The method we established has been applied to the therapeutic drug monitoring on cryptococcal-meningitis-infected patients who were given comedication of these two drugs.