Anabolic steroids are banned from use in food-producing animals in the European Union (Council Directive 96/22/EC). To control the zero-tolerance concept, an LC-MS/MS method for the screening and confirmation of most of the relevant natural and synthetic estrogenic and androgenic steroids in bovine and porcine blood plasma was developed and validated. The method permits confirmation and quantification of all analytes above a concentration of 0.65 µg L(-1). The validation was carried out 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 and different cartridge lots) were systematically varied on two levels.