In this study, the endolysosomal dynamics of H9 human embryonic stem cells (hESC) undergoing apicosome formation was examined using a spatial proteomics approach based on the engineered ascorbate peroxidase (APEX) 2, followed by mass spectrometry analysis. In detail, we generated H9 hESC line that express APEX2 fused to Podocalyxin in a doxycycline (DOX) inducible manner, which were then grown in the apicosome forming condition in the presence of DOX. At 30-hr timepoint, proximity biotinylation was performed using our previously established APEX2-labeling strategy, which was followed by TMT labeling and mass spectrometry analysis.