To investigate peroxisomal proliferator-activated receptor alpha (PPARalpha) signal responses in heart muscle, we performed LC-MS/MS-based proteomics analysis of heart muscle from rats given fenofibrate or clofibrate. Fenofibrate increased the expression of ACAA2, DECR1, and ECH1 consistent with activation of PPARalpha. Fenofibrate and clofibrate reduced the expression of 10 and 12 proteins, respectively with the expression of ACSL1, SLC25A4, A1BG, HADHA, ATP2A2, BDH1, ETFDH, HADHB, and CPT2 being reduced in common with both of fibrate-treated groups. The approach adopted in this study provides an efficient method for monitoring global changes in protein expression.