Isavuconazole (ISZ) is a newly available broad-spectrum triazole agent recently approved for the treatment of both invasive aspergillosis and mucormycosis. The aim of this study was to develop a simple and reliable method for therapeutic drug monitoring (TDM) of ISZ in human plasma samples. The method involves using a kit from ChromSystems intended for TDM of itraconazole (ITZ), posaconazole (PSZ), and voriconazole (VRZ) in serum/plasma for sample preparation and high-performance liquid chromatography, using fluorescence detection with emission and excitation wavelengths set to 261 and 366 nm, respectively. The assay was linear over the ISZ concentration range of 0.2 to 20.0 mg/liter, using a 0.1-ml sample volume. The inter- and intraday coefficients of variation were all below 3.7%, whereas the accuracies ranged from 95.0 to 106.2% and the mean extraction recovery was 91.9%. In addition, the method worked well using four different Vacutainer types, with six different healthy volunteers and under a number of relevant storage conditions. Finally, the ISZ detection could be seamlessly implemented in the TDM kit for VRZ, PSZ, and ITZ, enabling simultaneous detection of all four triazoles. This method proved to be simple, accurate, precise, and well suited for routine analysis work. It has been implemented in our laboratory for the simultaneous quantitative analysis of ISZ, VRZ, PSZ, and ITZ for TDM and pharmacokinetic research.