The integration of proximity labeling (PL) and advanced mass spectrometry-based proteomics is a robust framework for mapping protein–protein interaction (PPI) networks and local protein inventories in the crowded multimolecular environment of live cells. Over the last decade, numerous PL technologies such as biotin identification (BioID), ascorbate peroxidase (APEX) etc. using engineered enzymes or synthetic photocatalysts have been developed and successfully used in cell-based experiments. However, the application of such technologies beyond cultured cells, (i.e. in more complicated tissues or in vivo) remains challenging. In this review, we summarize the current issues in applying PL methods in vivo and highlight recent studies that could provide breakthroughs to overcome the existing limitations and expand the application of PL to tissues and in vivo.