Subcellular localization is a main determinant of protein function; however, a global view of cellular proteome organization remains relatively unexplored. We have developed a robust mass spectrometry-based analysis pipeline to generate a proteome-wide view of subcellular localization for proteins mapping to 12,418 individual genes across five cell lines. Based on more than 83,000 unique classifications and correlation profiling, we investigate the effect of alternative splicing and protein domains on localization, complex member co-localization, cell-type-specific localization, as well as protein relocalization after growth factor inhibition. Our analysis provides information about the cellular architecture and complexity of the spatial organization of the proteome; we show that the majority of proteins have a single main subcellular location, that alternative splicing rarely affects subcellular location, and that cell types are best distinguished by expression of proteins exposed to the surrounding environment. The resource is freely accessible via www.subcellbarcode.org. Display omitted •Robust method for proteome-wide subcellular localization analysis•Resource for subcellular location of 12,418 proteins in multiple cell lines•Analysis of protein domain- and variant-associated localization•Global analysis of protein relocalization driven by EGFR inhibition Orre et al. use mass spectrometry to map the subcellular localization of more than 12,000 proteins across several cell lines and present a method for proteome-wide relocalization analysis. This study provides information about the spatial organization of the proteome and an accessible resource for protein localization, subcellbarcode.org.