Shotgun proteomics can be applied to identify and study insect species in diverse research areas such as agriculture, forensics, biodiversity conservation, and food safety. In this chapter, we have provided a detailed protocol for shotgun proteomics analytical methods involving enzymatic digestion of insect proteins using trypsin, separation using high-performance liquid chromatography, and detection of separated peptides using high-resolution mass spectrometry. The protocol also covers the utilization of bioinformatics software for protein identification and spectral library building, proposing both proteomic database-dependent and independent methods. This chapter provides a valuable foundation for applying insect proteomics by discussing a step-by-step protocol.