Novel, economical, and eco-friendly UPLC method was optimized and validated for simultaneous determination of cefalexin (CFX) and preservative components namely, sodium benzoate (SB) and methylparaben (MP) in their dosage form, spiked human plasma, wastewater of poultry slaughter and in rabbit plasma using cefradine (CFR) as an internal standard (IS). Chromatographic system was optimized using design of experiments (DoE) such as Box-Behnken design (BBD) and response surface methodology (RSM) to improve the quality of the analytical methods, minimize the risk of method failure and provides a better solution for the defect results. Three input factors (independent variables) such as composition of methanol in the mobile phase, concentration of the phosphate buffer of the aqueous phase and its pH value were selected to study their effects on (dependent variables) as retention time, theoretical plate, and resolution. Isocratic chromatographic conditioning was created using mobile phase consisting of methanol: phosphate buffer (30mM) at pH 3.0 ± 0.1 (35:65, v/v), Agilent ZORBAX SB-C18 column (50 mm × 2.1 mm, 1.8 μm particle size) at flow rate 0.4 mL/min, injection volume 0.3 μL for RP-UPLC and UV detection at 230 nm; the retention time was 1.73 min for CFX, and column temperature was adjusted at 40ºC. The suggested method was validated as per the guidelines of the FDA for bioanalytical method validation and could be applied to quality control and laboratory prepared mixtures.