BACKGROUND: Current guidelines recommend intravenous thrombolysis (IVT) for acute ischaemic stroke (AIS) patients within 4.5 hour (h) of symptom onset. Our study aims to use proteomic biomarkers to identify AIS patients with an onset time within 4.5 h when the history is not clear. METHODS: We conducted a retrospective case-control study between June 2022 and July 2023 in Ningbo No. 2 Hospital, recruiting 30 AIS patients and 12 controls. Patients with AIS were grouped into early-onset (ES, symptom onset time ≤ 4.5 h, n = 16) and late-onset (LS, symptom onset 4.5-24 h, n = 14). Plasma proteome were identified using mass spectrometry. A stepwise analysis was conducted to screen for candidate proteins. Multiple logistic regression was used to construct various combinations. RESULTS: Here we show six proteins discriminate ES from LS, with the area under curve (AUC) ranging from 0.897 to 0.951. Protein 4.2 (EPB42) achieves the highest AUC of 0.951 (95% confidence interval 0.882-1), a specificity of 0.929 (0.714-1) and a sensitivity of 0.875 (0.750-1). Ten combinations are derived from these six proteins, of which EPB42 and Phosphatidylethanolamine-binding protein 1 achieve an AUC of 0.991 (0.970-1), a specificity of 0.929 (0.857-1), and a sensitivity of 1 (0.875-1) in differentiating ES from LS. CONCLUSIONS: The six proteins and their combinations show promise as molecular clocks for determining the onset time of AIS in patients whose symptom onset time are unknown, potentially increasing their chances of receiving effective IVT to improve stroke outcomes.