¹Department of Quality Assurance, Y. B. Chavan College of Pharmacy, Aurangabad, 431001, India DOI : 10.29228/jrp.475 The present study describes the principles of systematic Quality by design (QbD) approach for the development of RP-HPLC method for the quantification of Bupivacaine hydrochloride (BUP) in bulk and in-house developed nanostructured lipid carriers (NLCs). Initially analytical target profile (ATP), and critical analytical attributes (CAAs) were identified. Primary assessment studies were checked using Plackett-Burman Design. Further optimization studies were performed by applying Box-Behnken design. The Shimadzu C-18 column (250mm x 4.6mm i.d., 5μm particle size) was utilized for reversed-phase chromatographic separation with a mobile phase comprising a mixture of acetonitrile (ACN) and 0.1% ortho phosphoric acid (OPA) (pH 2.04) in 69.45:30.55 (% v/v). The flow rate was 0.805mL/min at a λ max of 214 nm and an injection volume of 12μL. The new developed method was validated according to the guidelines given by International Conference on Harmonization which revealed linearity between 25 to 80μg/ml and r2 = 0.999. The result of % RSD was 0.38 and 0.44 respectively for high degree of intraday and interday precision. As per the new method the LOD and LOQ is 0.900μg/ml and 2.72μg/ml, respectively. Further the validated method was also applied for the estimation of BUP in NLCs formulation, which showed no interference of any formulation excipients. The studies demonstrated that the new method is rapid, simple, selective, and reproducible for the estimation of pure drug and in-house developed NLCs. Keywords : Bupivacaine hydrochloride; Quality by design; Nanostructured lipid carriers; HPLC, Validation