Human Norwalk virus (HuNoVs), the most common etiological agents of acute gastroenteritis, are genetically diverse RNA viruses that frequently cause mass food poisoning internationally. In this study, we developed a parallel reaction monitoring (PRM) assay to simultaneously detect and identify representative peptides derived from VP1 in six different HuNoV genotypes using a high-resolution mass spectrometer. An optimized protocol was also used to detect and quantify VP1 protein in human stool specimens. This method is expected to become a new tool for studying the molecular epidemiology of HuNoV and to shed new light on targeted proteomics in clinical practice.