Great progresses have been made for generating in vitro pluripotent stem cell pancreatic β-like cells. However, the maturation stage of the cells still requires in vivo maturation to recreate the environmental niche. A deeper understanding of the factors promoting maturation of the cells is of great interest for clinical applications. We performed label-free mass spectrometry-based proteomic analysis of samples from a longitudinal study of differentiation of human-induced pluripotent stem cells towards glucose-responsive insulin producing cells.