Epitope tagging is a versatile approach to study diferent proteins using a well-defned and established methodology. To date, most epitope tags such as myc, HA, V5 and FLAG tags are recognized by antibodies, which limits their use to fxed cells, tissues or protein samples. Here we introduce a broadly applicable tagging strategy utilizing a short peptide tag (PepTag) which is specifcally recognized by a nanobody (PepNB). We demonstrated that the PepNB can be easily functionalized for immunoprecipitation or direct immunofuorescence staining of Pep-tagged proteins in vitro. For in cellulo studies we converted the PepNB into a fuorescently labeled Pep-chromobody (PepCB) which is functionally expressed in living cells. The addition of the small PepTag does not interfere with the examined structures in diferent cellular compartments and its detection with the PepCB enables optical antigen tracing in real time. By employing the phenomenon of antigen-mediated chromobody stabilization (AMCBS) using a turnover-accelerated PepCB we demonstrated that the system is suitable to visualize and quantify changes in Pep-tagged antigen concentration by quantitative livecell imaging. We expect that this novel tagging strategy ofers new opportunities to study the dynamic regulation of proteins, e.g. during cellular signaling, cell diferentiation, or upon drug action.