During food processing and storage, insult-induced crosslinks form between proteins, which profoundly influence the nutritional value and properties of food. One crosslink that forms with heating and basic pH, two typical insults used in food preparation and processing, is lysinoalanine. This crosslink has been shown to decrease levels of lysine, an essential amino acid, and reduce digestibility. In vivo studies suggest that it may be toxic. Despite the adverse effects of lysinoalanine, its formation and biological fate is not well understood. The typically low relative abundance and complex fragmentation pattern of crosslinked peptides compared to linear peptides, together with several other factors, makes investigating crosslinks in peptides challenging. To simplify the complex fragmentation patterns and narrow the potential number of analytes, we subjected two short synthetic peptides, each five amino acids long, to heat and alkaline treatment and identified a crosslink of lysinoalanine between the peptides. Using mass spectrometry we have characterised the fragmentation pattern of the model and identified a diagnostic ion for the lysionalanine crosslink. This diagnostic fragmentation pattern has the potential to be used to identify the location of lysinoalanine formation in proteins, which is not presently possible through standard or customised bioinformatics software. Knowing where lysinoalanine forms enables this structural aspect to be included in digestion models. These results also give clues to help us predict how altering food preparation procedures will influence formation of lysinoalanine and the opportunity to increase foods’ nutritional value.