Atazanavir (ATV) is widely used as anti-HIV agent with poor aqueous solubility which requires fabrication of novel drug delivery system to enhance therapeutic activity and safety. For this purpose, the quality by design (QbD) based ATV loaded nanostructured lipid carriers (NLCs) to address the challenges of bioavailability and its safety on oral administration. Herein, the main objective was to identify the influencing variables for the production of quality product. Considering this objective, quality target product profile (QTPP) was assigned and a systematic risk assessment study was performed to identify the critical material attributes (CMAs) and critical process parameter (CPP) having an influence on critical quality attributes (CQAs). Lipid concentrations, surfactant concentrations, and pressure of high-pressure homogenizer were identified as CMAs and CPP. ATV-NLCs were prepared by emulsification-high pressure homogenization method and further lyophilized to obtain solid-state NLCs. The effect of formulation variables (CMAs and CPP) on responses like particle size (Y1), polydispersity index (Y2), and zeta potential (Y3) was observed by central composite rotatable design (CCRD). The data were statistically evaluated by ANOVA for confirmation of a significant level (P<0.05). The optimal conditions of NLCs were obtained by generating design space and desirability value. The lyophilized ATV-NLCs were characterized by DSC, PXRD, and FT-IR analysis. The morphology of NLCs was revealed by TEM and FESEM. In vitro study suggested a sustained release pattern of drug (92.37±1.03 %) with a mechanism of Korsmeyer-Peppas model (r2 =0.925, and n=0.63). In vivo evaluation in Wistar rats showed significantly higher (p<0.001) plasma drug concentration of ATV-NLCs as compared to ATV-suspension using chylomicron flow block model. The relative bioavailability of ATV-NLCs was obtained to be 2.54 folds. Thus, a safe and promising drug targeting system was successfully developed to improve bioavailability and avoiding first-pass effect ensures to circumvent the acute-toxicity of liver.