This study describes the development of a novel assay for SARS-CoV-2 identification using LC-MS/MS analysis. A multi-step procedure for the rational down-selection of a set of markers has leaded to the discovery of six SARS-CoV-2 specific and sensitive markers, enabling the reliable identification of the virus. A rapid and simple assay was developed, successfully applied to clinical nasopharyngeal samples. The assay may potentially serve as a complementary approach for SARS-CoV-2 identification.