Purpose In this study, first, second, third, and fourth-order derivative spectrophotometric methods utilizing the peak—zero (P—O) and peak-peak (P—P) techniques of measurement were developed for the determination of levofloxacin, norfloxacin, and moxifloxacin. These methods were applied to their combined pharmaceutical dosage form or individually for levofloxacin, norfloxacin, and moxifloxacin. Methods Linearity was established in the concentration range of 2–20 μg/mL. The procedures are simple, quick, and precise. The developed methods are sensitive, accurate, and cost-effective, demonstrating excellent correlation coefficients (R2 = 0.9998) and mean recovery values ranging from 99.20% to 100.08%, indicating a high level of precision. Results The developed approach was effectively employed to determine the levofloxacin, norfloxacin, and moxifloxacin content in commercially available pharmaceutical dosages. Conclusions Statistical analysis and recovery tests confirmed the method’s linearity and accuracy. The results suggest that this method can be utilized for routine analysis in both bulk and commercial formulations. The simplicity, accuracy, and cost-effectiveness of the developed methods make them valuable for pharmaceutical analysis.