A new, simple, inexpensive, and rapid 96-well plate UV spectrophotometric method was developed and validated for the quantification of compound 48/80 (C48/80) associated with particles. C48/80 was quantified at 570 nm after reaction with acetaldehyde and sodium nitroprusside in an alkaline solution (pH 9.6). The method was validated according to the recommendations of the ICH Guidelines for specificity, linearity, range, accuracy, precision, and detection and quantification limits (DL and QL). All the validation parameters were assessed in three different solvents, i.e., deionized water, blank matrix of chitosan nanoparticles, and blank matrix of chitosan/alginate nanoparticles. The method was found to be linear in the concentration range of 5 to 160 μg/ml (R2 > 0.9994). Intraday and interday precision was adequate, with relative standard deviation lower than those given by the Horwitz equation. The mean recoveries of C48/80 from spiked samples ranged between 98.1% and 105.9% for calibration curves done with the blank matrices and between 89.3% and 103.3% for calibration curves done with water, respectively. The DL were lower than 1.01 μg/ml and the QL were lower than 3.30 μg/ml. The results showed that the developed method is sensitive, linear, precise, and accurate for its intended use, with the additional advantages of being cost-effective and time-effective, allowing the use of small-volume samples, and the simultaneous analysis of a large number of samples. The proposed method was already successfully applied to evaluate the loading efficacy of C48/80 chitosan-based nanoparticles and can be easily applied during the development of other C48/80-based formulations.