<p>The exact estimation of protein content is required in the biochemical, medical, and food sectors. The current technique, Kjeldahl procedure is time-consuming and uses hazardous reagents; therefore, the search for an easier, safer and time-saving method is of paramount importance. This study aimed to develop and validate a novel spectrophotometric method for the quantification of protein in animal-derived food products by measuring the absorbance of ammonium ions released after acid digestion. Bovine Serum Albumin (BSA) was used as a standard reference protein. Acid digestion of BSA was performed and the samples were scanned using UV–Vis spectrophotometry (200–800 nm), revealing a λ_max at 260 ± 1 nm. A linear calibration graph of BSA was established for protein quantification, and the results were compared with those determined by the Kjeldahl method and other spectrophotometric assays. The method was then applied to various animal food matrices. The proposed method achieved a λ_max at 260 ± 1&#xa0;nm and demonstrated &gt; 90% protein recovery across diverse food samples, including fresh and frozen veal, chicken, egg albumin and yolk, and beef hotdogs. Statistical analysis (<i>p</i> &gt; 0.01) showed no significant difference between this method and the Kjeldahl and other spectrophotometric methods. The principal UV-absorbing compound was conclusively identified, after distillation and collection of the digested proteins in three different acid solutions (boric, hydrochloric, and sulfuric) without a dye indicator. The collected solutions were scanned revealing a λ_max of 255 ± 2 nm, conclusively representing ammonium ions as the primary absorbing compound, since the solutions are solely composed of ammonium ions and the acids. This method provides a rapid, accurate, and reliable alternative to the Kjeldahl method for protein quantification in animal products, bypassing the other stages used in Kjeldahl, and offers comparable analytical performance.</p>