Organoids have emerged as a revolutionary ex vivo 3D culture model for medicinal research. Despite their promising applications, the detailed compositions of organoids, particularly their proteome and glycoproteome, remain underexplored, with limited studies comparing these profiles to those of the original tissues. Additionally, obtaining high-quality mass spectrometric data from the small sample amounts of organoids poses a challenge. In this study, we successfully generated proteomic and glycoproteomic profiles using only 1-5 μg of protein from hepatocellular carcinoma (HCC) organoids. Our global proteomics analysis identified 2,099 proteins, highlighting key metabolic processes such as RNA metabolism, nucleobase-containing small molecule metabolic processes, and carbon metabolism, which are reflective of the primary metabolic functions of liver tissue. Glycoproteomic analysis revealed 41 glycoproteins from 159 glycopeptides, including significant therapeutic targets and biomarkers for HCC, such as EGFR and AFP. Comparative analysis of glycan compositions between HCC tissue and HCC-derived organoids demonstrated their similarity, suggesting that organoids retain the essential characteristics of the actual tissues.