Quality control for human induced pluripotent stem cells (hiPSCs) is important for efficient and stable production of hiPSC-derived cell therapy products to be used for transplantation. During cell culture, hiPSCs spontaneously undergo morphological changes and lose pluripotent properties. Such cells are termed deviated cells, which are altered from the undifferentiated state of hiPSCs, and express the early differentiation marker stage-specific embryonic antigen 1 (SSEA-1). In this study, we searched for soluble SSEA-1(+) glycoproteins secreted from deviated cells generated by culturing hiPSCs in cell culture medium containing heat-inactivated supplements. Glycoproteins obtained from cell culture supernatants of SSEA-1(+) deviated cells were enriched by an O-glycan binding lectin and blotted with anti-SSEA-1 antibody. A single protein band at >250 kDa specifically detected by anti-SSEA-1 antibody was identified as fibronectin (FN) by LC-MS/MS analysis and immunoprecipitation combined with western blotting, indicating that FN is a carrier protein of SSEA-1. We then constructed a sandwich enzyme-linked immunosorbent assay to detect SSEA-1(+) FN secreted from deviated cells. This FN-SSEA-1 test proved to be both sensitive and specific, allowing for non-destructive detection of SSEA-1(+) deviated cells within mixed cell population, with a lower limit of detection of 100 cells/mL. The developed assay may provide a standard technology for quality control of hiPSCs used for regenerative medicine. (C) 2020 Elsevier Inc. All rights reserved.