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 × 104 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.