Background and Aims Multisystem inflammatory syndrome in children (MIS-C) is a rare inflammatory condition, occurring 1-2 months after SARS-CoV-2 infection in children. The clinical features of MIS-C are nonspecific. Since early treatment is of prognostic value, clinicians are faced with the challenge to recognize patients timely. However, lacking a specific test, diagnosing MIS-C remains largely based on clinical expertise. Enrichment of Vβ21.3+ T-cell receptors is a frequent finding in MIS-C. We aimed to assess the value of whole blood (WB) Vβ21.3 staining as a diagnostic test for MIS-C. Methods EDTA blood was obtained in healthy controls (HCs) and patients fulfilling the WHO MIS-C case definition (before immunomodulatory treatment). WB was stained, lysed and fixed within 2h of sampling. By flow cytometry, the relative frequency of Vβ21.3+ T-cells (CD3/CD4/CD8) was assessed as well as activation markers (HLA-DR). Results Four patients and five HCs were included. The Vβ21.3+ T-cell proportions were increased in one case (3.6-5.6% of T-cells), a 12yo girl presenting typical MIS-C and responding rapidly with first-line treatment. Although all other patients fulfilled the MIS-C case definition at inclusion, their further disease course led to alternative diagnoses, including Streptococcus pyogenes sepsis, haemophagocytic lymphohistiocytosis and metabolic cardiomyopathy. These non-MIS-C patients and HCs showed only 1.0-2.6% Vβ21.3+ T-cells. Additionally, in MIS-C 37.7% of Vβ21.3+ lymphocytes were HLA-DR+, while only 0.1-11% in HCs and non-MIS-C patients. Conclusions We provide proof-of-concept that Vβ21.3 staining can be used as a rapid diagnostic test to distinguish MIS-C from other inflammatory diseases. Its sensitivity and specificity should be assessed in larger prospective studies.