In Brief The interior of the cell is molecularly crowded. Its compartmentalization within organelles enables the regulation of biochemical processes and allows multifunctionality of proteins and RNAs. Subcellular information can thus give insights into the function of these biomolecules. Multiple techniques to measure such information have been established, with ever-increasing throughput and sensitivity. These techniques are covered in this review, and demonstrating their application is providing valuable insights into cellular biology, such as aiding our understanding of single-cell heterogeneity and posttranslational modifications.