The objective of the Current research was to apply Quality by Design approach for development of more accurate, precise, specific and robust RP-HPLC method for estimation of spironolactone in its tablet dosage form and Human Plasma. The Full 3 level Factorial design was applied on mobile phase Composition & pH of buffer system as considering CQAs against dependent variables viz. retention time, peak asymmetry, theoretical plates. By using design space numerical, graphical optimization on retention time, peak asymmetry, and theoretical plates and the optimum chromatographic conditions were chosen. Optimized analytical method consisted Acetonitrile: Ammonium Formate (60:40v/v) as mobile phase, pH 3.5, flow rate 1ml/min, a wavelength 237 nm. Protein precipitation technique is used in preparing samples for bioanalysis. Spironolactone was extracted from human plasma using acetonitrile as a precipitating agent, and the supernatant was then injected. Spironolactone was eluted with retention time of 5.235 min and peak area of 920547. With a correlation coefficient of 0.999, Spironolactone had an excellent linear relationship in the range of 5-30 g/ml. RSD % for intraday, interday, and repeatability were determined to be 1.69%, 1.87%, and 0.59% respectively. The limit for detection and quantification was determined to be 0.40g/ml and 1.23g/ml. The parameters used for method validation were within the ICH-recommended range. The suggested approach is working well for the best analysis of pharmaceutical dosage forms of Spironolactone in bulk and was effectively used in a pharmacokinetic investigation