In this study, we engineered a micro-well duct-on-chip platform to generate defined 3D aggregates from hiPSC-derived PPs and subsequently induce differentiation toward PDLOs. Time-resolved scRNA-seq combined with cleared immunofluorescence imaging provided a deep understanding of in vitro ductal cell type differentiation. By defining the emergent cell types at each stage of differentiation based on their gene expression profiles and organoid structures, we provide a precise cell-by-cell description of the in vitro differentiation trajectory. Transcriptional data of PDLOs were complemented by their proteome and secretome data, allowing the identification and validation of prognostic cancer marker. Thus, we show the applicability of hiPSC-derived PDLOs-on-chip for future ductal disease modeling.