OBJECTIVES: The aim was to develop and validate a LC-MS/MS assay to determine antiretrovirals in human plasma for routine therapeutic drug mon-itoring. METHODS: The selectivity, sensitivity, linearity, accuracy, precision, recovery, matrix effect, stability and dilution integrity and carry-over were validated ac-cording to EMA and FDA standards. RESULTS: For accuracy and precision, the highest overall bias was 11.3% at LLOQ of both lopinavir and saquinavir. The highest overall CV was 15.6% at the LLOQ of darunavir. Storage stability at 4°C, 20–25°C and 10°C had a maximum CV of 13.2% at low QC level (0.2 mg/L) for saquinavir. Freeze-thaw stability had a maximum overall bias of 7.4% at low QC level (0.8 mg/L) for tipranavir. Selectivity and specificity showed no interfering peaks of more than 20% of the LLOQ. CONCLUSIONS: The bioanalytical method is suitable for both TDM in stan-dard care and clinical studies. Werf T, Touw D, Alffenaar JW. Develop-ment and validation of a bioanalytical method for the simultaneous determi-nation of 14 antiretroviral drugs using liquid chromatography-tandem mass spectrometry. J Appl Bioanal 4(2), 37-50 (2018).