A sensitive and robust liquid chromatography-tandem mass spectrometry (LC-MS/MS) method allowing the rapid screening and confirmation of thyreostatic drugs in bovine blood plasma was developed and validated according to Commission Decision 2002/657/EC, chapter 3.1.3 "alternative validation", by applying a matrix-comprehensive in-house validation concept. Decision limit CCα, detection capability CCβ, recovery, repeatability, within-laboratory reproducibility and the uncertainty of measurement were calculated. Furthermore, a factorial effect analysis was carried out to identify factors that have a significant influence on the method. Factors considered to be relevant for the method in routine analysis (e.g. operator, storage duration of the extracts before measurement, different cartridge lots and duration of sample preparation) were systematically varied on two levels during the validation study. Subsequently, the extent to which these factors influence the measurement results of the individual analytes was examined.