Horseshoe crabs are among the most studied invertebrates due to their unique, innate immune system and biological processes. The metabolomics study was conducted on lipopolysaccharide (LPS)-stimulated and non-stimulated hemocytes isolated from the Malaysian Tachypleus gigas and Carcinoscorpius rotundicauda. LC-TOF-MS, multivariate analyses, principal component analysis (PCA), and partial least squares-discriminant analysis (PLS-DA) were included in this study to profile the metabolites. A total of 37 metabolites were identified to be differentially abundant and were selected based on VIP > 1. However, of the 37 putative metabolites, only 23 were found to be significant with ANOVA at p