Reliable and quantifiable high-resolution protein localization is critical for understanding protein function. However, the time required to clone and characterize any protein of interest is a significant bottleneck, especially for electron microscopy (EM). We present a modular system for enzyme-based protein tagging that allows for improved speed and sampling for analysis of subcellular protein distributions using existing clone libraries to EM-resolution. We demonstrate that we can target a modified soybean ascorbate peroxidase (APEX) to any GFP-tagged protein of interest by engineering a GFP-binding peptide (GBP) directly to the APEX-tag. We demonstrate that APEX-GBP (1) significantly reduces the time required to characterize subcellular protein distributions of whole libraries to less than 3 days, (2) provides remarkable high-resolution localization of proteins to organelle subdomains, and (3) allows EM localization of GFP-tagged proteins, including proteins expressed at endogenous levels, in vivo by crossing existing GFP-tagged transgenic zebrafish lines with APEX-GBP transgenic lines. Display omitted •APEX-GBP allows for high-resolution subcellular protein distribution analyses•APEX-GBP allows for quantitative and volumetric assessment of protein distribution•APEX-GBP is conducive to rapid screening of GFP-tagged clone libraries by EM•APEX-GBP is a sensitive and inducible system for protein localization in vivo Rapid screening with electron microscopy (EM)-based analysis has been difficult. Here, Ariotti et al. generate and characterize a system for fast, sensitive, and reliable localization of GFP-tagged proteins to nanometer resolution. APEX-GBP zebrafish lines are also crossed with GFP-tagged lines to rapidly determine whole organism protein distribution at EM resolution.