Information of protein localization on subcellular level is important to map and characterize the proteome and to better understand cellular functions of proteins. Here, we report on a pilot study of 466 proteins in three human cell lines aimed to allow large-scale confocal microscopy analysis using protein-specific antibodies. Approximately 3000 high resolution images were generated and more than 80% of the analyzed proteins could be classified in one or multiple subcellular compartment(s). The localizations of the proteins showed, in many cases, good agreement with the Gene Ontology localization prediction model. This is the first large-scale antibody-based study to localize proteins into subcellular compartments using antibodies and confocal microscopy. The results suggest that this approach might be a valuable tool in conjunction with predictive models for protein localization.