The elucidation of the metabolic fate of prohibited substances is crucial for the abuse detection. The human hepatocyte cell line HepG2 can be used to study biotransformation. In order to improve this in vitro model system, we compared the HepG2 spheroid generation using three different techniques: a forced floating, a scaffold-free and a scaffold-based method. We characterized the spheroids with regard to the expression levels of the proliferation marker Mki67, the liver-specific marker albumin and biotransformation enzymes. Moreover, the metandienone metabolite pattern was comparatively analysed by high-performance liquid chromatography mass spectrometry. With all three techniques, HepG2 spheroids were generated showing a degree of differentiation. The forced floating method resulted in very large spheroids (1 mm in diameter) showing signs of necrosis in the centre and a very low metandienone conversion rate. The spheroids formed by the two other techniques were comparable in size with 0.5 mm in diameter on average. Among the three different 3D cultivation methods, the HepG2 spheroids formed on Matrigel® as extracellular matrix were the most promising regarding biotransformation studies on anabolic androgenic steroids. Prospectively, HepG2 spheroids are a promising in vitro model system to study multidrug setups, drug-drug interactions and the biotransformation of other substance classes.