ABSTRACTMirabegron, a drug for treatment of overactive bladder, has low water solubility and bioavailability. Researchers used quality by design to develop and characterize second‐generation lipid nanocarriers for mirabegron, aiming to provide spatially and temporally sustained drug release. Numerous analytical methodologies for assessing mirabegron in pharmaceutical and biological fluids use chemical solvents in the mobile phase, potentially impacting aquatic ecosystems and the environment. The issue was addressed with a microwave‐assisted spectrofluorimetric technique, environmentally benign solvents, and a fluorescent probe known as 4‐chloro‐7‐nitrobenzofuran. Researchers used screening design and response‐surface methodology to minimize organic waste. The suggested technique satisfied ICH Q2 (R2) and M10 validation standards. This approach described the in vivo pharmacokinetics and in vitro drug release of mirabegron‐loaded lipid nano‐carriers. We anticipated the chemical reaction pathways that derivatized mirabegron with 4‐chloro‐7‐nitrobenzofuran by the analysis of mass spectra. We assessed the proposed method's environmental effect and sustainability compared to current methods using the RGB model, an analytical greenness calculator, and green analytical process index software. The proposed mirabegron estimate method was user‐friendly for analysts, cost‐effective, sensitive, robust, and environmentally sustainable, adhering to sustainable analytical practices and reducing ecological consequences.