<p>How proteins localize to specific compartments, function in coordination with other biomolecules and, ultimately, contribute to diverse cellular activities are crucial questions in cell biology. Complicating the answers to these questions are multilocalizing and multifunctional proteins, whose impact on the cell depends on both spatial and temporal contexts. Therefore, contextualizing protein functions based on their subcellular localization is necessary to fully understand cell behaviours. Recent advances in instrumentation and protein labelling techniques are rapidly increasing the availability of tools, technologies and applications that measure and control protein localization and compartment-specific function. In this Review, we first discuss microscopy, mass spectrometry-based correlation profiling and proximity labelling methods that assign localizations to proteins, ranging from cellular compartments to protein–protein interactions. We next examine the available tools for manipulating protein localization and measuring the effects of these manipulations, including localization tags and bifunctional molecules. For each technology, we assess the strengths and weaknesses that ultimately determine their usefulness. We conclude with an outlook on future technological advances in the field of spatial subcellular proteomics and their potential implications for cell biology and clinical applications.</p>