This study describes, for the first, the development and validation of a novel high-throughput micorwell spectrophotometric assay for determination of the quinolone antibiotic rosoxacin (ROS) in its bulk and capsules. The assay involved the reaction between ROS and sodium nitroprusside (SNP) forming a red-colored product exhibiting maximum absorption peak (λmax) at 455 nm. The reaction was carried out in 96-microwell plate and the absorbance of the colored-product was measured by microwell plate absorbance reader at 455 nm. The variables affecting the reaction were carefully investigated using the microwell format and the optimum conditions under were established. Under the optimized conditions, a linear relationship with good correlation coefficient (0.9992) was found between the absorbance of the ROS-SNP chromogen and ROS concentration in the range of 15-150 μg/mL. The limits of detection and quantification were 3.5 and 11.6 μg/mL, respectively. The assay showed high precision as the values of relative standard deviations (RSD) did not exceed 2%. The proposed assay was successfully applied to the determination of ROS in its capsules; the label claim percentages were 102.81±1.43%. The results were compared favorably with those of a reference pre-validated method. The proposed assay is practical and valuable in terms of its routine application in determination of ROS in its bulk and capsules in pharmaceutical quality control laboratories.