Understanding the dynamic protein–protein interactions (PPIs) is fundamental to deciphering cellular signaling pathways and biological processes. Traditional methods for identifying PPIs, such as yeast two-hybrid and co-immunoprecipitation, often lack spatiotemporal resolution and may miss weak or transient interactions. Proximity-dependent biotinylation (PDB) coupled with mass spectrometry (MS) has emerged as a powerful technique to map the spatial proteome in living cells. Among various engineered biotin ligases, Turbidite-Identification (TurboID) stands out for its unprecedented catalytic efficiency, enabling rapid biotinylation of neighboring proteins within minutes. This chapter provides a detailed, step-by-step protocol for conducting a TurboID-based proximity labeling experiment to identify potential interacting proteins of a target protein of interest.