Withanolides are a class of ergostane natural products found in plants of family Solanaceae. Plants of this family are used in traditional medicine in Asia and South America. Recently, a series of 17β-hydroxy withanolides were identified from high-throughput screens as inhibitors of androgen-induced changes in gene expression of prostate cancer cells. Therefore, these compounds may have important applications as new therapies against prostate cancer. We have devised a synthetic route to members of this family and their analogues which allows stereoselective introduction of C14, C17 and C20 hydroxyl groups in separate steps. This will allow preparation of differentially hydroxylated analogues so as to identify which contributes to the potency and thus gain a better understanding of the SAR of this class of bioactive molecules. As part of this we have shown that the stereochemical outcome of the epoxidation of Δ 14-15 cholestanes with m-CPBA is controlled by the steric bulk of a C17 substituent. When the C17 is in the β configuration, the epoxide is formed on the α face, whereas if the C17 is trigonal (flat) or the substituent is in the α configuration, the epoxide is formed on the β face. The presence of a hydroxyl substituent at C20 does not influence the stereochemical outcome of the epoxidation. We have successfully introduced aldehyde functionality to the lateral side chain 14 hydroxyl compound. This aldehyde compound is a key intermediate from which many of the withanolides can be made. We have also investigated the introduction of a hydroxyl at the C18 as an entry into the physachenolides. Finally, we have carried out an assessment of the potency of the synthesised compounds against hormone-insensitive prostate cancer cell line, PC-3.