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Aim and Objectives: In the present investigation dual agents i.e., hesperidin and diazepam loaded polymeric nanoparticles (NPs) were formulated by nanoprecipitation method and optimized using three-level factorial design. Methods: The developed NPs were optimized keeping poly (lactic-co-glycolic) acid (PLGA), poloxamer amount as independent process variable and z-average, percentage drug entrapment as a dependent response. The optimized NP was subjected to in vitro drug release study to investigate drug release mechanism from NP. Cell viability assay was performed on Vero cell line to confi rm the safety of NP. Results: Drug loaded NP showed z-average in the range of 189-307 d.nm with percentage drug entrapment for diazepam and hesperidin 62-89% and 68-92%, respectively. In vitro drug release studies showed controlled drug release behavior was observed from polymeric NP across dialysis membrane compared to aqueous drug solution. Cell viability assay showed drug dependent cytotoxicity on Vero cell line, however, polymeric NP showed less cytotoxicity compared with aqueous drug solution. [ABSTRACT FROM AUTHOR]