A method for sensitive analysis of 19 anabolic steroids (AS) in animal oil using enhanced matrix removal lipid (EMR-Lipid) cleanup and ultrahigh performance liquid chromatography-tandem mass spectrometry (UHPLC-MS/MS) was developed. Oil samples were extracted with 20 mL of acetonitrile aqueous solution and purified using EMR-Lipid cartridges. The eluent was evaporated to dryness under nitrogen and analyzed by UHPLC-MS/MS using 0.1% formic acid–acetonitrile and 0.1% formic acid–water solutions as the mobile phase via gradient elution. The method effectively removed unwanted matrix co-extractives better than other extraction cleanup techniques while still delivering acceptable recovery results for most of the AS. The established quantification method showed AS recovery in the range of 72.9–110.7% with good precision (relative standard deviation < 15%).