Cartilage damage may eventually lead to osteoarthritis because it is difficult to repair. Human-induced pluripotent stem cell (iPSC)-derived chondrocytes may potentially be used to treat cartilage damage, but the tumorigenicity of iPSCs is a major concern for their application in regenerative medicine. Many glycoconjugates serve as stem cell markers, and glycosphingolipids (GSLs) including H type 1 antigen (Fucα1-2Galβ1-3GlcNAc) have been expressed on the surface of iPSCs. The purpose of the present study was to investigate whether GSL-glycome analysis is useful for quality control of residual iPSCs in chondrocytes. We performed GSL-glycome analysis of undifferentiated iPSCs in chondrocytes by combining glycoblotting and aminolysis-sialic acid linkage-specific alkylamidation (SALSA) method, enabling the detection of small quantities of iPSC-specific GSL-glycans from 5 × 10 4 cells. Furthermore, we estimated the residual amount of iPSCs using R-17F antibody, which possesses cytotoxic activity toward iPSCs that is dependent on the Lacto-N-fucopentaose I (LNFP I) of GSL. Moreover, we could detect a small number of LNFP I during mesenchymal stem cells (MSCs) differentiation from iPSCs. This is the first demonstration that GSL-glycome analysis is useful for detecting undifferentiated iPSCs, and can thereby support safe regenerative medicine. a regenerative medicine for cartilage repair [1]. ACI has advantages over existing methods in which the injured part is covered with hyaline cartilage, but there remain issues such as the sacrifice of healthy cartilage, the need for two operations, and difficulty in acquiring a sufficient number of cells due to low proliferation ability. In addition, Roberts et al. reported that more than half of the cartilage tissue repaired with ACI were predominantly fibrocartilage composed of type I collagen and IIA procollagen, which were different from normal hyaline cartilage composed of Type II collagen [2].Human-induced pluripotent stem cells (iPSCs) proliferate indefinitely in an undifferentiated state, and their pluripotency makes them capable of differentiating into any tissue in the human body [3]. Regenerative medicine using iPSC-derived cells has potential for repairing defective cartilage. Indeed, iPSCs can be differentiated into chondrogenic lineages [4], and iPSC-derived cartilaginous particles can repair articular cartilage defects in mini-pigs. However, the tumorigenicity and dedifferentiation of iPSCs are barriers that must be overcome before the true potential of iPSCs in regenerative medicine can be realized. Since even a small number of residual iPSCs may form teratomas [5], it is a prerequisite for clinical application that the final products contain no iPSCs. To evaluate whether iPSCs are included, fluorescence labeling and observation with a fluorescence microscope was applied [6], as has labeling and evaluation by flow cytometry [7], but it is difficult to confirm that all iPSCs are actually labeled using these techniques. Moreover, undifferentiated cell removal methods...