Traditional approaches, such as microscopic inspection and PCR, are inadequate for accurately identifying the gelatin types and animal sources of leather. The current investigation aimed to establish an Ultra-High-Performance Liquid Chromatography/Electrospray Ionization-Quadrupole Time-of-Flight-High Resolution Mass Spectrometry UHPLC/positive ESI-Q-TOF-HRMS technique for identifying variations in the amino acid sequences of type I collagen hydrolysate. Gelatin, primarily obtained from bovine sources, has found extensive application in various food and pharmaceutical applications. To assure the compliance of food products with halal rules, accurate analytical procedures are very much necessary. A specific marker peptide for bovine gelatins was chosen in this research to establish a system for multiple reaction monitoring using UHPLC/positive ESI-qTOF-HRMS. The current work aimed to optimize the UHPLC/ESI-Q-TOF-HRMS method for the identification of gelatin types and collagen hydrolysates. This was achieved by controlled enzymatic digestion of leather white pickled shaving samples and then matching their LC/ESI-Q-TOF-HRMS output data (Rt, XIC, monoisotopic masses of the molecular and/or selective fragment ions of some peptide markers) with a respective library database. Two bacterial strains, namely BFW (5,7), were isolated from fish wastes and applied to white shavings that were supplied by the Egyptian leather industry. These strains have demonstrated their ability to produce collagenase, which is a potent enzyme that facilitates the controlled hydrolysis of collagen. This enzymatic process gives gelatin of high quality, which can be utilized as a valuable resource in industrial applications. The present investigation involved the identification of a wide range of collagen marker peptides in bovine collagen hydrolysates using a sequential process of chromatography. The current UHPLC/positive ESI-Q-TOF-HRMS findings indicate that more than 2000 and 4000 peptides were tentatively identified in both investigated collagen hydrolysate samples, i.e., BFW5 and BFW7. According to their peptide sequence, the structure of the major peptides was identified from higher to lower MW as GEPGPTGIQGPPGPAGEEGKR and GPAGPQGPR, respectively, for the BFW5 sample. Also, for the BFW7 sample, the higher-to-lower MW of major peptides were identified as GAPGDKGEAGPSGPAGPT and LAGPPGESGR, respectively. The above results showed that the peptides isolated in this study were identified and characterized as collagen marker peptides from bovine collagen hydrolysates. The quantification technique was thus developed using the three most frequently occurring peptides in the digested bovine gelatin, namely GFOGADGVAGPK, GETGPAGROGEVGPOGPOGPAGEK, and GFOGSOGNIGPAGK. When it comes to detecting bovine gelatin, these collagen marker peptides are more unique. When utilized in conjunction with HPLC and mass spectrometry, this technique serves as a precise and very sensitive quantitative approach for the detection of bovine gelatins. Therefore, this approach can be employed to authenticate the halal status of gelatin.