Expansion of the available repertoire of reagents for visualization and manipulation of proteins will help understand their function. Short epitope tags installed on proteins of interest and recognized by existing binders such as nanobodies facilitate protein studies by obviating the need to isolate new antibodies directed against them. Nanobodies have several advantages over conventional antibodies, as they can be expressed and used as tools for visualization and manipulation of proteins in vivo. Here, we combine the advantages of short epitopes (NanoTags) and nanobodies specific for them by characterizing two short (<15 aa) tags, 127D01 and VHH05, which are high-affinity targets of nanobodies. We demonstrate that these NanoTags and the nanobodies that recognize them can be used in Drosophila for in vivo protein detection and re-localization, direct and indirect immunofluorescence, immunoblotting, and immunoprecipitation. We further show that CRISPR-mediated gene targeting provides a straightforward approach to tagging endogenous proteins with the NanoTags. Single copies of the NanoTags, regardless of their location, suffice for detection. This versatile and validated toolbox of tags and nanobodies will serve as a resource for a wide array of applications, including functional studies in Drosophila and beyond.Competing Interest StatementThe authors have declared no competing interest.