The article reports on the development of an efficient, robust and sensitive HPLC-DAD method for the simultaneous determination of five fluoroquinolone-based antimicrobial drugs, namely ciprofloxacin, moxifloxacin, norfloxacin, ofloxacin and pefloxacin in both aquatic and tablet formulations. The robustness of the high-performance liquid chromatography with diode-array detection (HPLC-DAD) method has been evaluated through the concepts of quality-by-design (QbD) and full factorial design of experiments (DoEs), using a Minitab 17 statistical tool. The proposed method offers sequential separation with well-defined peak shape and resolution, and has also been evaluated by following international council for harmonization (ICH) pharmaceutical guidelines. A linear signal response has been achieved for the target fluoroquinolones (FQ) drugs in the concentration range of 45-20,000 ng/mL, with an average correlation coefficient (r2) value of 0.9997, and a data precision and accuracy range of 99.3-100.9%, with an RSD value of ≤0.95%, for hexaplicate measurements. The methodology offers superior sensitivity for the target FQ drugs, with the limit of detection (LD) range of 10-25 ng/mL, and the limit of quantification (LQ) range of 51-86 ng/mL, respectively. Using the proposed method, the article carries the first of its kind report in studying the degradation profile monitoring and drug assay determination in tablet formulations and under various physiological buffer stress conditions, for pharmaceutical validation.