In the present study, supercritical fluid (carbon dioxide) chromatographic methods were developed and validated for the quantitative assay determination of two cancer therapeutic substances, fulvestrant and azacitidine, using a UPC2 system. Fulvestrant was separated from six potential impurities, while impurities of azacitidine were separated using a 150 mm × 4.6 mm, I.D., a chiral column and 5 μm, particle size. Both drugs were analysed simultaneously in a single sequence using multiple channels in the system and respective methods. These new methods were validated for their intended purpose in accordance with the current ICH guidelines. The method exhibited excellent intra- and inter-day precision. A precision with RSD 1% and 1.6% was achieved for fulvestrant and azacitidine, respectively. A linear relationship r² > 0.999 was achieved between the concentration and detector response over a range of 25–150% of the target concentrations for both compounds. The two compounds were well quantified from their unspecified impurities obtained from stress studies. The method can be employed for routine quality control testing and stability analysis.