There is no established method for measuring human hepatic triglyceride (TG) lipase (HTGL) concentration in serum. In this study, we developed new monoclonal Abs (MoAbs) (9A1 mouse MoAb and 141A1 rat MoAb) that react with HTGL both in serum and in postheparin plasma (PHP) and established a novel ELISA system for measuring serum HTGL and PHP-HTGL concentrations. To confirm the specificity of MoAbs, we performed immunoprecipitation-immunoblotting analysis. Both 9A1 mouse MoAb and 141A1 rat MoAb were able to immunoprecipitate not only recombinant HTGL and PHP-HTGL but also serum HTGL, demonstrating that HTGL exists in serum obtained without heparin injection. This method yielded intra- and interassay coefficients of variation of <6% and showed no cross-reactivity with LPL or endothelial lipase. In clinical analysis on 42 male subjects with coronary artery disease, there were strong positive correlations of serum HTGL concentration to PHP-HTGL concentration (r = 0.727, P < 0.01). Serum HTGL concentrations showed positive correlations to serum TGs (r = 0.314, P < 0.05) and alanine aminotransferase (r = 0.406, P < 0.01), and tendencies toward positive correlations to LDL cholesterol, small dense LDL, and γGTP. These results suggest that this new ELISA method for measuring serum HTGL is applicable in daily clinical practice.