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Biomarkers that indicate the presence or severity of organ damage caused by diseases and toxicities are useful diagnostic tools. Metabolomics platforms using chromatography coupled with mass spectrometry (MS) have been widely used for biomarker screening. In this study, we aimed to establish a novel metabolomics platform using ion chromatography coupled with MS (IC-MS) for human biofluids. We found that ethylenediaminetetraacetic acid (EDTA) plasma is not suitable for IC-MS metabolomics platforms because of the desensitization of MS. IC-MS enabled detection of 131 polar metabolites in human serum and urine from healthy volunteers. Pathway analysis demonstrated that the metabolites detectable using our platform were composed of a broad spectrum of organic acids with carboxylic moieties. These metabolites were significantly associated with pathways such as the tricarboxylic acid (TCA) cycle; glyoxylate and dicarboxylate metabolism; alanine, aspartate, and glutamate metabolism; butanoate metabolism; and the pentose phosphate pathway. Moreover, comparison of serum and urine samples showed that four metabolites (4-hydroxybutyric acid, aspartic acid, lactic acid, and γ-glutamyl glutamine) were abundant in serum, whereas 62 metabolites, including phosphoric acid, vanillylmandelic acid, and N-tiglylglycine, were abundant in urine. In addition, allantoin and uric acid were abundant in male serum, whereas no gender-associated differences were found for polar metabolites in urine. Our results demonstrate that the present established IC-MS metabolomics platform can be applied for analysis of human serum and urine as well as detection of a broad spectrum of polar metabolites in human biofluids.