Ischemic penumbra is the main therapeutic target for acute ischemic stroke. The aim in this study is to investigate the potential serum biomarkers of penumbra that could fulfill a complementary role in the acute stroke clinical decision-making process. An established focal cerebral ischemia model is applied in rats. Using isobaric tags for relative and absolute quantitation combined with liquid chromatography-tandem mass spectrometry, the global expression profiles of proteins in ischemic penumbra tissue and serum from rats subjected to different ischemic times are identified and quantified. Candidate biomarkers are screened out with bioinformatics analysis and further validated by western blotting. Herein, a total of 4568 proteins in the penumbra and 1915 proteins in the serum are identified. Two proteins related to the penumbra, RHOA, and CDC42, are screened out through an integrative analysis. The expression levels of RHOA and CDC42 in the penumbra and serum gradually increase synchronously with the prolonged ischemia time. The study provides the results of a proteomic analysis to identify serum biomarkers of the penumbra. Upregulation of RHOA and CDC42 may be useful for the early assessment of ischemic penumbra and could serve as potential serum biomarkers. © 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.