The chapter describes the bioconversion of phytosterols to androstenedione (AD) with Mycobacterium spp. in shake flasks and fermenters, as well as LC-MS based methods for analysis of phytosterols and steroid products.Phytosterols are derived as a by-product of vegetable oil refining and of manufacture of wood pulp. Phytosterols contain the same four-ring nucleus as steroids, and may be converted to high-value steroids by removing the side chain at C17 and minor changes at other sites in the ring structure.Many bacteria, including Mycobacterium spp., are able to degrade phytosterols. Mutants of Mycobacterium spp. unable of ring cleavage can, when growing on phytosterols, accumulate the steroid intermediates androstenedione (AD) and/or androstadienedione (ADD).The practical challenge with microbial conversion of phytosterols to steroids is that both the substrate and the product are virtually insoluble in water. In addition, some steroids, notably ADD, may be toxic to cells.Two main strategies have been employed to overcome this challenge: the use of two-phase systems, and the addition of chemically modified cyclodextrins. The latter method is used here.Defined cultivation and bioconversion media for both shake flask and fermenter are given, as well as suggestions to minimize the practical problems caused by the water-insoluble phytosterol. Sampling, sample extraction, and quantification of substrates and products using LC-MS analysis are described.