The objective of the study was to evaluate blood metabolites as indicators of susceptibility to subacute ruminal acidosis (SARA) in mid-lactation Holstein cows. Seventy-eight lactating dairy cows (DIM = 103 ± 27; BW = 630 ± 77 kg) were fed a high-concentrate diet (65% of DM) during a 24-d study. Cows were classified according to rumenocentesis pH, measured 4 h after the morning feeding on the last day of the study, as tolerant (pH ≥6.0; n = 26), marginal (5.8 ≤ pH <6; n = 21), and susceptible (pH <5.8; n = 31). Cows were also classified according to reticuloruminal pH, measured during the final 5 consecutive days for 14 cows via indwelling sensors, as susceptible if duration of an acidotic condition (pH <5.8) exceeded 330 min/d (n = 9) and tolerant if the acidotic condition persisted <330 min/d (n = 5). Blood was sampled 4 h after morning feeding on d 18 of the study and immediately serum were harvested. Data were analyzed using Mixed Procedures of SAS with a model included the fixed effects of SARA susceptibility (rumenocentesis-based or reticuloruminal-based) and the random effect of parity. The classification based on rumenocentesis pH showed serum activity of aspartate aminotransferase (AST) was greater (P < 0.03) in susceptible cows (70.7 U/L) than tolerant (56.6 U/L) and marginal (59.9 U/L) cows. Also, the blood urea nitrogen (BUN) concentration was greater (P = 0.01) with SARA susceptible cows (13.6 mg/dL) than marginal (12.2 mg/dL) and tolerant (12.5 mg/dL) cows. Similarly, blood albumin concentration was greater for SARA susceptible cows than for marginal and tolerant cows (P < 0.02). The classification based on reticuloruminal pH also resulted in a trend of greater AST (P = 0.08) activity and greater BUN concertation (P < 0.05) in the blood of susceptible cows. Glucose, triglyceride, cholesterol, globulin, alkaline phosphates and serum amyloid A did not differ among the different susceptibility classes. Among blood metabolites measured, serum concentration of AST was in the strongest correlation with rumenocentesis pH (n = 78; r = −0.32; P = 0.01) and milk fat percentage (n = 78; r = −0.23; P = 0.04) and therefor it may be a good indicator of acidosis susceptibility in mid-lactation dairy cows.