Tuberculosis (Tb) is a serious worldwide threat to human health killing 1.4 million people every year. Mycobacterium tuberculosis (M. tuberculosis), the causative agent for Tb, has infected 30% of the world's population. The emergence of multidrug, extensively drug, and total drug resistant organisms (MDR/XDR/TDR TB) creates an urgent demand to develop new drugs with novel mechanisms to be effective against both sensitive and drug-resistant strains of M. tuberculosis. The cholesterol metabolism pathway in M. tuberculosis is important for mycobacterial survival in the host macrophage. We proposed that cholesterol serves as a starting material for biosynthesis of steroid-derived immunomodulators that down-regulate the host immune response in the intracellular environment. Therefore, identifying the metabolites, which are hypothesized to enable M. tuberculosis to modulate and evade the host immune response, can help elucidate the cholesterol metabolic pathways, presenting an attractive target for new drugs. To determine whether M. tuberculosis produces immunomodulatory compounds that act as agonists or antagonists of nuclear receptors, our objective is to use bioassays to identify and isolate metabolites biosynthesized from cholesterol by Mycobacterium Smegmatis (M. smegmatis). We set up the procedure for purification and analysis of low-density lipoprotein-cholesterol (LDL-cholesterol). By measuring the changes in fluorescence polarization, we also set up a screening assay for metabolites from M. smegmatis grown on LDL-cholesterol against glucocorticoid receptor.