Subacute ruminal acidosis (SARA) resulting from excessive grain feeding to dairy cows is accompanied by an immune response and has been associated with impaired liver function and blood composition. We, therefore, investigated the proteome in blood plasma to discover potential biomarkers for SARA. Four lactating Danish Holstein cows were used in a study that included a 4-week control period followed by a one-week SARA challenge period. During the control period, all cows received a total mixed ration (174 g crude protein/kg DM; 192 g starch/kg DM; 6.28 MJ NE_L/kg DM) ad libitum. During the SARA period, 40% of the ration was gradually substituted with grain pellets (50:50 wheat:barley) within 3 d to induce SARA. This SARA induction diet was then fed for another 4 d. Rumen pH was monitored with indwelling pH probes to verify that cows had rumen pH lower than 5.6 for 3 h daily during the SARA induction. Blood samples were obtained from the jugular vein at 7 h post-feeding on d 4 before SARA induction and on the second of 4 SARA-induction days. The blood plasma samples were then processed for the enrichment of low molecular weight serum proteins using acetonitrile precipitation before mass spectrometry-based proteomic analysis on a nano-RPLC-MS/MS system. The analysis identified 14 high-confidence proteins with significant quantitative differences between control and SARA periods as defined by MS intensity, of which 9 were potential differentially expressed (PDE) proteins LASP1, TMSB4, and the precursors of KNG1, APOE, SERPINA3–8, CRP, SERPINF2, SERPIND1 and CATHL4 reportedly involved in acute phase response, cytoskeletal proteins, blood coagulation, cholesterol transport, liver disease, and innate immune defense molecules. Among these PDE proteins, LASP1 and the precursors of APOE, SERPINA3–8, SERPINF2, SERPIND1 and CATHL4 were upregulated and others were downregulated (P < 0.05). These results suggest that it may be possible to identify proteins that are differentially expressed during SARA. Studies are ongoing to validate the possible candidates and identify additional proteins for the understanding of ethological concepts of SARA.