We present an effective non-targeted workflow for screening hazardous proteins in alternative protein-based foods (APBFs) that includes a protocol for efficient sample preparation, an optimised liquid chromatography with tandem mass spectrometry (LC-MS/MS) approach, and a data analytics pipeline with a comprehensive hazardous protein database. In addition, we developed a targeted MS assay for validating our screening results that confirmed the presence of some of selected hazardous proteins, and showed the quantification capability of this targeted assay at femtomole sensitivity.