The incidence of invasive fungal infections has been increasing due to the widespread clinical use of high-dose chemotherapy drugs and immunosuppressive agents. Fungal infections are associated with higher mortality than other infections due to their rapid progression. To achieve therapeutic efficacy and simultaneously avoid toxicity, therapeutic drug monitoring (TDM) has increasingly been adopted for antifungal agents with narrow therapeutic indices. Several methods have been developed for the quantitation of antimycotics in biological fluids. High-performance liquid chromatography based on MS/MS and UV detection are the most widely used. To maximize the clinical benefit of TDM, accurate quantitative results with short turnaround times are required.