Liquid chromatography linked with mass spectrometry (LC–MS/MS) was used to analyse gelatin from four different species after a trypsin digest. Using chemometric software to analyse the data it was possible to find peptide fragments that were specific to each species of gelatin: porcine, bovine, chicken or fish. Identification of these peptides was challenging due to the destructive nature of gelatin manufacture. The untargeted workflow method developed allowed identification of 21 unknown gelatin samples with 100% accuracy. Fish gelatin is made from a large range of different species that do not share a common differentiating protein but it was shown that the protein from a parasitic bacteria could be used to identify fish gelatin.