Fisetin (FS) is a phyto-flavonoid with antioxidant, neuroprotective, and anticancer properties. A bioanalytical method was developed and validated to estimate FS levels in rat plasma using reverse phase ultra-fast liquid chromatography with a C-18 reverse phase column. Quercetin (Qu) served as an internal standard. The mobile phase consisted of acetonitrile and orthophosphoric acid (0.2% v/v) in a 30:70 v/v ratio, with a flow rate of 1 mL/min, and detection was performed at a wavelength of 362 nm. Protein precipitation was employed to extract the drug from plasma samples. The retention times for FS and Qu were 5.6 min and 10.3 min, respectively. The method demonstrated linearity in the range of 2-10 ng/mL with a regression coefficient (r²) of 0.9994. Validation followed the ICH Q2 (R1) guidelines, with percentage recovery between 98-100%, indicating accuracy. The percentage relative standard deviation was below 2%, showing precision. The limit of detection (LOD) and limit of quantification (LOQ) were 0.03 ng/mL and 0.08 ng/mL, respectively. The method proved robust, showing no significant response changes with variations in flow rate and mobile phase composition. These results indicate that the developed method meets all validation criteria and is suitable for estimating fisetin in rat plasma.