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Objectives: Polymeric nanoparticles are a promising novel drug delivery system and have advantages in cancer therapy. Etoposide is an anticancer agent that is used in the treatment of a variety of malignancies. The aim of the present study was to prepare and evaluate novel polymeric nanoparticles containing etoposide. Materials and Methods: A 3² full factorial design was used to study the effect of Eudragit EPO and Pluronic F-68 on the characterization of nanoparticle suspensions. The polymeric nanoparticles were prepared by nanoprecipitation technique. The prepared nanoparticles was evaluated by percentage yield, drug polymer compatibility using fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetric (DSC) analysis, drug content, entrapment efficiency, zeta potential, particle size, scanning electron microscopy, X-ray diffraction, in vitro drug release studies, kinetic modeling, stability studies, and in vivo animal studies. Response surface plots were studied, which were generated using PCP dissolution software. Results: Scanning electron microscopic studies confirmed their porous structure with a number of nanochannels. The FTIR spectra showed the stable character of etoposide in a mixture of polymers and revealed the absence of drug-polymer interactions. The DSC study revealed that the drug was involved in complexation with nanoparticles. The average particle size of etoposide nanoparticles was in the range of 114.4 nm to 136.7 nm. The zeta potential values were attained to ensure good stability of nanosuspensions. In vitro release of the drug from nanoparticles follows the Peppas model and showed controlled release behavior for a period of 24 h. The optimized nanoparticles were subjected to stability studies at 4°C in a refrigerator and the most suitable temperature for storage of etoposide nanoparticles found. The average targeting efficiency of drugloaded nanoparticles was 41.88±0.030% of the injected dose in the liver, 25.66±0.320% in the spleen 13.82±0.090% in the lungs, 4.52±0.300% in the kidney, and 4.18±0.490% in the brain. Conclusion: Etoposide loaded nanoparticles was found to be effective in sustained release. [ABSTRACT FROM AUTHOR]