Resveratrol and Gefitinib are adjunct therapies for various cancers; however, both have been limited by low solubility, low cellular uptake, and bioavailability issues. As a result, this research aimed to develop an accurate, precise, selective, and sensitive reverse-phase high-performance liquid chromatography (RP-HPLC) method to simultaneously determine both compounds in nanoformulation and Glioma cells. The phenomenex luna C8 column, a mobile phase (80: 20 ratios of acetonitrile: 200 mM ammonium acetate) with a flow rate of 1 mL. min⁻¹, 40 ± 0.2 °C as a column temperature, and the injection volume was 20 μl were selected as optimized chromatographic conditions. Retention time (RT) of resveratrol (1.80 min) and gefitinib (2.56 min) were identified using an optimized analytical method and detected at 345 nm (isosbestic point). The approach was proven to be specific for resveratrol and gefitinib analysis in the existence of PHLNPs, precise (RSD 2 %), and accurate (>90 %). The simultaneous analytical method was successfully developed to identify percentage drug entrapment efficiency (% DEE), % drug loading (% DL) of resveratrol and gefitinib in PHLNPs, and secondary estimates of in-vitro drug release profile and percentage cellular uptake studies. The in-vitro results revealed that the developed analytical method could simultaneously detect and quantify these drugs in other nanoformulations and in-vivo studies.