Dairy-derived endogenous peptides, as important bioactive components in dairy products, demonstrate significant research value and application potential in fields such as food science, nutrition, and biomedicine. However, their precise analysis is constrained by several bottlenecks such as complex matrix interference and difficulties in detecting low-abundance peptides. Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) provides an efficient solution to overcome these bottlenecks by leveraging its advantages of high throughput, high sensitivity, and rapid analysis. This article outlines the technical principle and strengths of MALDI-TOF MS, along with the strategies used to address its technical limitations, and highlights its applications in areas including dairy traceability and authentication, processing monitoring, functional peptide identification, and quality and safety control. Integration of MALDI-TOF MS with other technologies and algorithmic models significantly improves peptide profiling analysis, showing broader application in the dairy industry. It provides stronger technical support for the nutrition, functionality, and safety of dairy products and also opens up new avenues for research in related fields.