Human hepatic organoids (hHOs) are regarded as physiologically relevant in vitro platforms to evaluate hepatotoxicity, a critical step in drug development, but their applications are currently limited by the lack of qualified and standardized evaluation markers. In this study, by leveraging the established reference measurement system of amino acids (AAs), we propose 12 new biomarkers for drug-induced hepatotoxicity evaluation in human induced pluripotent stem cell-derived hHOs. Two orthogonal analytical methods for AAs were developed and validated based on isotope dilution mass spectrometry. Four AAs (aspartic acid, arginine, glutamine, and phenylalanine) and eight ratios of two designated AAs in the media of hHOs showed reliable alteration by drug treatment, which was confirmed by differentiating between hepatotoxic and nonhepatotoxic drugs. The superiorities of AA-based toxicity evaluation using the media of hHOs are as follows: (i) ability to use media only, without direct damage to or consumption of the organoids, (ii) ability to measure and compare quantities of AAs through a standardized reference measurement system rather than nonstandardized cell viability indicators, and (iii) no requirement for further data normalization in the case of the AA ratios. The AA analysis-based results demonstrate the reliability and potential of the proposed biomarkers as not only straightforward indicators of drug-induced hepatotoxicity but also absolutely comparable measures as a step toward standardization based on the AA reference measurement system.