A design of experiments (DoE)-based quality by design (QbD) method was used to develop a stabilityindexing assay method in high performance thin layer chromatography (HPTLC) for the kinetic study of fluoxetine. This research proposes to use an environmentally friendly HPTLC technique based on Analytical Quality by Design (AQbD) to determine environmental friendliness. According to the literature, there is currently no published article on this method. A stationary phase consisting of TLC plates (Merck) pre-coated with silica gel 60F254 on aluminum sheets was used. A mobile phase of acetone: water with 1% orthophosphoric acid (8.5:1.5 v/v) was used. Central Composite Design (CCD) provides the opportunity to optimize the HPTLC separation and determine the parameters that have the greatest influence and those that have interactions. 20 optimized experimental runs were performed, including organic solvent content (A), saturation time (B), and driving distance (C). These 3 factors were evaluated for robustness, showing an insignificant effect on retention. The Rf value was 0.72±0.07, and the calibration curves were linear in the range of 0.3-5 mcg/point with regression coefficients r2 of 0.9987 for fluoxetine HCl. In the intraday and interday precision study, the percent RSD was 0.26 and 0.28, respectively. Fluoxetine was found to be highly susceptible to alkaline and acid hydrolysis compared to oxidation. All system suitability parameters were validated and were within the range specified in ICH guidelines. The novel method was also evaluated using four different approaches, including other evaluation methods: NEMI, GAPI, AGMS, AGREE, and it was found to be environmentally friendly.