For the simultaneous measurement of Ethinylestradiol (EE) and Drospirenone (DP) in fixed-dose combination hormones tablets, a reverse-phase high-performance liquid chromatographic (RP-HPLC) method was developed. A specific, precise and accurate RP-HPLC method was developed and validated to analyse the drugs in rat plasma. The fluorescence detection for EE was made at λ= 200-310 nm and Ultraviolet-visible (UV/Vis) detection for DP was made at 270 nm. The typical EE and DP retention times were 4.19 and 5.30 minutes, respectively. The limit of detection (LOD) and limit of detection (LOQ) for EE were 0.121 and 0.282µg/mL and LOD and LOQ for DP were 2.23 and 7.697µg/mL respectively. The regression coefficient (r2) of EE and DP were 0.9937 and 0.9913 respectively. Precision's relative standard deviation (RSD) was less than 5%. The analyte recoveries of both drugs stayed within 95% of each other. All other validation parameters adhered to ICH standards. Throughout the analytical process, the analyte was stable. The advantages of the method developed include stability under different conditions and a low limit of quantification that was in micrograms. Its applicability was confirmed by the analysis of EE and DP levels in plasma samples in a designed pharmacokinetic study in rats after oral administration.