This dataset contains the raw data of mass spectrometry from a STUPPIT pull-down experiment investigating the intermediary proteins between two non-interacting proteins. We developed STUPPIT (Split-TurboID and PUP-IT based Protein Identification Tool), a novel method to biotinylate intermediary proteins of two non-interacting proteins through a two-step enzymatic reaction. STUPPIT were validated using proteomics targeting YAP1&β-actin (Subproject IPX0013559002, using STUPPIT all-in-plasmid method, 3 repeats), YAP1&β-actin and YAP1&MOB1A (IPX0013559004,using stably cell line STUPPIT approach I, 2 repeats ) as shown in each subgroup. New intermediary proteins enriched by STUPPIT between β-catenin and SMAD4, key components of the Wnt and BMP signaling pathways, were also subjected to proteomics (Subproject IPX0013559003, 3 repeats). In conclusion, STUPPIT represents a powerful tool for labeling and capturing intermediary proteins between non-interacting partners, offering new insights into protein-protein interactions and advancing signal transduction research.