This project investigates the dynamic changes in global protein expression and phosphorylation modifications during the in vitro luteinization of human granulosa cells. Granulosa cells were cultured and harvested at three distinct time points: 0 hrs, 48 hrs, and 96 hrs. Protein extracts were subjected to tryptic digestion and analyzed by high-resolution LC-MS/MS to generate both comprehensive proteomic and phosphoproteomic datasets. This time-series profiling aims to decipher the molecular signaling pathways and regulatory networks governing granulosa cell luteinization, including steroidogenesis and cellular differentiation. Bioinformatic analysis of this time-series dataset identified the MAPK signaling pathway as a prominently regulated hub during luteinization progression. In the subsequent validation phase, granulosa cells were treated with U0126, a specific MEK1/2 inhibitor, under identical culture conditions, and samples at matched time points were subjected to the same proteomic and phosphoproteomic workflows. Comparative analysis between control and U0126-treated datasets will systematically characterize MAPK-dependent regulatory networks during in vitro luteinization.