Two most commonly used preparative methods, reverse phase evaporation and ethanol injection were employed to prepare cationic liposomes composed of Etoposide API, DMPG-Na polymer and Cholesterol binder, respectively. To overcome the hindrances of the reported HPLC analytical method in pharmacopeia which requires more time in preparation for solvent and also its bit tedious; we have developed and validated a simple method which will be applicable to detect and quantify actual drug in formulation as well as it can applied for pharmacokinetics study. The resulting formulations were evaluated through morphology observation, particle size and zeta potential analysis, % entrapment efficiency and % drug loading. The results showed that liposomes prepared by ethanol injection method were of best quality and stability, with promising results. However ETNLE 5 shows best results i.e. particle size 197.3±0.21nm, polydispersity index 0.340±0.051%, and zeta potential of about -12.7±1.266mV. Entrapment efficiency 81.78± 0.78% and drug loading 89.62±2.53% is the highest as compared to all other batches. % In-vitro Drug release study showed 15% and 21% of drug was released in the first five minutes with a cumulative drug release of 58% and 78% for ETNLE 5 formulation at pH 1.2 and pH 6.4 respectively. Stability study of optimized batch showed no significant changes in evaluation parameters. Cell viability study on A-549 cells by MTT assay clarified cancer cells are inhibited by 200 µM equivalent etoposide liposomes as compared to 64.88% of free drug. These findings clarified the effect of preparative methods on performance of cationic liposome, as well as formulation factors on entrapment efficiency, and will provide important methodological reference for further study of liposomes carriers for drug delivery to tumor penetration.