Background: Glypican-3 (GPC3) is expressed in most of hepatocellular carcinoma (HCC) and GPC3 immunohistochemical staining is widely used in the clinical setting, but it has not been recommended as a blood biomarker, mainly due to its heterogeneous nature and the lack of established assay system. Here, we developed and evaluated the basic performance of fully automated GPC3 immunoassay kits which detect the full-length or the N-terminal fragments. We analyzed the molecular forms of GPC3 in HCC serum and evaluated the diagnostic performance of GPC3 and other biomarkers. Methods: We examined the analytical performance of the GPC3 kits. Then, the automated GPC3 assays were compared with an established ELISA kit. Afterwards, we determined the clinical cutoff of GPC3 and compared its diagnostic performance to alpha-fetoprotein (AFP) and protein induced by vitamin K absence or antagonist-II (PIVKA-II) using 180 serum samples from clinically diagnosed patients. Results: GPC3 assays showed good analytical performance. The level of GPC3 in HCC was higher than recorded in healthy or other liver diseases’ sera. The AUC of GPC3 was 0.90, whereas the AUCs of AFP and PIVKA-II were 0.89 and 0.76, respectively. Conclusion: Automated GPC3 assays with stable performance against GPC3 in screening HCC have been established and the diagnostic accuracy of GPC3 was as good as AFP.