Since cardiac and skeletal myotoxicity affect the development of drug candidates, it is important to detect their toxicity at an early stage of drug development. For that purpose, in this study, the usefulness of several cardiac and skeletal myotoxic biomarkers in blood were evaluated using two rat models treated intraperitoneally with an acetylcholinesterase inhibitor carbofuran (CAF) or a synthetic catecholamine isoproterenol (ISO). The biomarkers assayed were fatty acid binding protein 3 (Fabp3), myosin light chain 1 (MLC1), cardiac troponin I (cTnI), cardiac troponin T (cTnT), aspartate transaminase (AST), lactate dehydrogenase (LDH) and creatine kinase (CK). CAF and ISO treatment of rats induced greater increases in the levels of Fabp3, MLC1, cTnI and cTnT than in the levels of AST, LDH and CK. A kinetic analysis indicated that the levels of all of the biomarkers had returned to the basal level by 24h after drug administration. Pathological examination revealed lesions in the heart, mainly at the left ventricle and septum, in both CAF- and ISO-treated rats. CAF-treated rats showed widespread lesions of skeletal muscle that were independent of muscle fiber type, while in ISO-treated rats locoregional lesions were observed only in slow twitch muscle. Receiver operating characteristic curve analysis of the sensitivity of the tested biomarkers indicated that MLC1 and cTnT were the most effective biomarkers of cardiotoxicity. For skeletal myotoxicity, Fabp3 and MLC1 were the most effective biomarkers based on the specific tissue distribution of these proteins. Conversely, the rapid blood clearance of these markers should be taken into account when considering the use of these biomarkers.