The aim of this project was to develop and validate an isotope-dilution liquid chromatography high resolution mass spectrometry (LC-HRMS) method for the quantification of the 11 most widely used systemic antimycotics and to study whether HRMS is a feasible alternative for therapeutic drug monitoring (TDM) when compared to tandem MS (MS/MS) technology. After protein precipitation, followed by automated online sample clean-up the analytes were separated within 4 min on a C18 column using an acetonitrile-water gradient. Eleven antimycotics, namely 5-flucytosine, amphotericin B, anidulafungin, fluconazole, isavuconazole, itraconazole, ketoconazole, micafungin, OH-itraconazole, posaconazole and voriconazole were finally quantified in full MS scan mode using positive electrospray ionization (ESI +) with a mass range fromm/z 110-1300 using HRMS. The method was comprehensively validated on the basis of the European Medicines Agengy (EMA) method validation protocol using commercially available IVD kit components. Good linear relationship between peak area responses and drug concentrations (R2 > 0.995) and excellent selectivity were observed for all antimycotics in this study. Inaccuracy and imprecision of all quality controls were consistently below ± 12.6% and ± 8.1%, respectively. Quantification results were in agreement with an IVD LC-MS/MS method. HRMS was shown to be suitable for TDM of small molecules when compared to tandem mass spectrometry. The novel HRMS method is quickly installed and may be a robust and reliable tool for routine TDM of antimycotics in clinical laboratories. Copyright © 2019 Elsevier B.V. All rights reserved.