Estramustine phosphate has been used for over 20 years in the treatment of advanced prostate cancer. Recent reports of promising results from clinical combination studies, and neoadjuvant studies, have caused a renewed interest in this relatively old drug. After oral or intravenous administration, estramustine phosphate is metabolised to four main metabolites: estramustine, estromustine, estradiol and estrone. Due to the potential biological activity of all these estramustine phosphate metabolites, it is necessary to analyse the parent compound and all four metabolites in plasma, tissues and tumours. Consequently, various methods have been developed for the bioanalysis of these compounds. In this review various methods are described for the analysis of estramustine phosphate and its metabolites in biological fluids and tissues. Methods described included RIA, TLC, HPLC, HPLC-MS, GC and GC-MS.