A multi-analyte, multi-matrix method was developed for the routine determination of steroids in animal tissues (skin, meat and fat). After addition of internal standards and sample pre-treatment, the analytes of interest were extracted from the matrix with unmodified supercritical CO2 and trapped directly on an alumina sorbent placed in the extraction vessel (in-line trapping under supercritical conditions). After extraction, alkaline hydrolysis was performed and the analytes were derivatised. The samples were then analysed by gas chromatography-mass spectrometry. The limit of detection for the different matrix–analyte combinations was 2 µg kg–1 (for melengestrol acetate 5 µg kg–1), the repeatability ranged from 4 to 42% (n = 9) and the reproducibility ranged from 2 to 39% (n = 3).