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This study aimed to design, optimize and validate an RP\u2010HPLC method for quantifying both drugs using an analytical quality by design (AQbD) approach. An analytical target profile (ATP) was defined, and risk assessment using an Ishikawa diagram identified mobile phase composition, flow rate and column temperature as critical parameters. Method optimization was performed using a Box\u2013Behnken Design, evaluating effects on retention times, peak areas, tailing factors and resolution. Optimal conditions included a 90:10 methanol:aqueous mobile phase, 0.5 mL\/min flow rate and 35\u00b0C column temperature. The method was validated per ICH Q2(R1) guidelines, demonstrating excellent sensitivity (LOD 0.69\u20130.29 \u03bcg\/mL), accuracy (95%\u2013105% recovery) and precision (RSD &lt;\u20092%). The method was successfully applied for drug estimation in liposomal formulations, ex\u00a0vivo permeation, cytotoxicity and cellular uptake studies. Liposomal gels showed high entrapment efficiency, enhanced drug release, permeation and cellular uptake. Analytical Eco\u2010Scale assessment confirmed excellent green performance.<\/jats:p>","DOI":"10.1002\/bmc.70393","type":"journal-article","created":{"date-parts":[[2026,3,8]],"date-time":"2026-03-08T18:55:46Z","timestamp":1772996146000},"update-policy":"https:\/\/doi.org\/10.1002\/crossmark_policy","source":"Crossref","is-referenced-by-count":2,"title":["Quality by Design\u2013Based RP\u2010HPLC Method for Simultaneous Estimation of 4\u2010Hydroxytamoxifen and Thymoquinone in Liposomal Formulation"],"prefix":"10.1002","volume":"40","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-0585-6329","authenticated-orcid":false,"given":"Pavithra\u00a0Pradeep","family":"Prabhu","sequence":"first","affiliation":[{"name":"Nitte (Deemed to Be University), Department of Pharmacognosy NGSM Institute of Pharmaceutical Sciences (NGSMIPS)  Mangalore Karnataka 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