Proximity labeling (PL) with TurboID provides a powerful alternative to traditional protein interaction mapping methods, allowing the capture of both stable and transient interactions in living cells. Here, we describe a standardized and optimized protocol for generating high-confidence proximity proteomes in Trypanosoma cruzi using a novel vector system, pTcTurboID. This vector allows the stable and regulatable expression of TurboID fusion proteins, ensuring minimal bait expression to preserve physiological function while maintaining sufficient biotinylation activity for protein detection. The protocol comprises eight steps, including the generation of spatial control and bait lineages, optimization of biotinylation conditions, and refinement of the purification process with magnetic streptavidin beads. Key features include the use of compartment-specific spatial controls to minimize nonspecific background and statistical analysis to identify true interactors. Our methodology has been validated with nuclear and cytoplasmic baits, yielding reproducible proximity interactomes. This protocol provides an efficient, reproducible, and robust framework for proximity proteomics in T. cruzi.