AbstractThe fight against hand, foot, and mouth disease (HFMD) remains an arduous challenge without existing point‐of‐care (POC) diagnostic platforms for accurate diagnosis and prompt case quarantine. Hence, the purpose of this salivary biomarker discovery study is to set the fundamentals for the realization of POC diagnostics for HFMD. Whole salivary proteome profiling was performed on the saliva obtained from children with HFMD and healthy children, using a reductive dimethylation chemical labeling method coupled with high‐resolution mass spectrometry‐based quantitative proteomics technology. We identified 19 upregulated (fold change = 1.5–5.8) and 51 downregulated proteins (fold change = 0.1–0.6) in the saliva samples of HFMD patients in comparison to that of healthy volunteers. Four upregulated protein candidates were selected for dot blot‐based validation assay, based on novelty as biomarkers and exclusions in oral diseases and cancers. Salivary legumain was validated in the Singapore (n = 43 healthy, 28 HFMD cases) and Taiwan (n = 60 healthy, 47 HFMD cases) cohorts with an area under the receiver operating characteristic curve of 0.7583 and 0.8028, respectively. This study demonstrates the feasibility of a broad‐spectrum HFMD POC diagnostic test based on legumain, a virus‐specific host systemic signature, in saliva.