A novel, simple reversed-phase high-performance liquid chromatographic (RP-HPLC) analytical method was developed and validated for the quantitative determination of asenapine from various nanoemulsion components during pre-formulation screening. The developed method was validated according to ICH Q2 (R1) guidelines. The developed and validated method was precisely and accurately quantified asenapine in various oils, surfactants and co-surfactants. The separation of asenapine was carried out on Hypersil BDS C18, 250×4.6mm, 5μm particle size column using methanol: acetonitrile (90:10) as mobile phase with a flow rate of 1mL.min⁻¹. Measurement at 270nm for the concentration range of 5 to 50μg.mL⁻¹ of the analyte was found to be linear with the determination coefficient (r²) of 0.999 as calculated by the least square regression method. The validated method was sensitive with LOD of 10.0ng.mL⁻¹ and LOQ of 30.0ng.mL⁻¹. Further, the method was precise and accurate, where the intraday and interday precision values were ranged from 0.70–0.95 and 0.36–0.95, respectively with the corresponding accuracy were ranged from 98.80–100.63 and 98.36–100.63. This developed and validated RP-HPLC method for asenapine was applied in the quantitative determination and screening of various oils, surfactants, and co-surfactants during the development of the asenapine maleate nanoemulsion.