eng Oriental Scientific Publishing Company Biosciences Biotechnology Research Asia 0973-1245 2026-09-09 23 3 59677 article Akanksha Kailas Nirmal 1 Mayur Shivaji Bhosale 1 Rahul Dnyaneshwar Khaire 2 Sanket Keshav Tambe 1 Shubham Popat Mankar 1 Shubham Balasaheb Mhaske 1 Department of Pharmaceutical Chemistry, Savitribai Phule Pune University, Pravara Rural College of Pharmacy, Pune, India Department of Pharmaceutical Chemistry, Savitribai Phule Pune University, PRES’s College of Pharmacy (For Women), Pune, India Reliable analytical methods are essential for the quality control of Lemborexant in pharmaceutical formulations. The present study aimed to develop and validate a simple, rapid, and robust Reverse Phase High-Performance Liquid Chromatography (RP-HPLC) method for the quantitative estimation of Lemborexant in bulk drug and tablet dosage forms using a Quality by Design (QbD) approach. Critical method parameters were systematically optimized using Design Expert® software (Version 13) to ensure consistent chromatographic performance. Separation was achieved on a Kromasil C18 column (250 mm × 4.6 mm, 5 µm) using a mobile phase consisting of acetonitrile (70:30, v/v) at a flow rate of 1.0 mL/min. Detection was carried out at 222 nm with an injection volume of 20 µL and a column temperature of 35°C. Under the optimized conditions, Lemborexant exhibited a retention time of 3.97 min. The developed method demonstrated excellent linearity over the concentration range of 10–30 µg/mL (R² = 0.9998). Validation performed according to ICH guidelines confirmed the method’s accuracy (mean recovery 99.89%), precision (%RSD < 2%), sensitivity (LOD 0.30 µg/mL and LOQ 0.90 µg/mL), and robustness. The results indicate that the proposed QbD-based RP-HPLC method is reliable, accurate, precise, and suitable for routine quantitative analysis and quality control of Lemborexant in bulk drug and pharmaceutical dosage forms. https://www.biotech-asia.org/vol23no3/quality-by-design-based-rp-hplc-method-development-and-validation-for-quantification-of-lemborexant/ Analytical Method Validation; Box–Behnken Design; Lemborexant; Quality by Design; RP-HPLC