Since its discovery, the high affinity interaction between biotin and avidin has formed the basis of numerous tools and techniques in molecular biology and biochemistry. The post-translational modifications of cellular proteins via conjugation of ubiquitin (Ub) and ubiquitin-like (UbL) proteins contribute to crucial cellular processes, such as protein homeostasis and the DNA damage response, yet they are challenging to study due to their dynamic nature, scarcity, and sensitivity to removal by proteases. Understanding how the Ub/UbL-modified proteome changes during development or in response to environmental insults or pathological conditions may yield new biomarkers or identify new drug targets. The use of biotin-based technologies for Ub/UbL studies is relatively new, but has already contributed to the identification of new substrates and promises much more. In this review, we focus on two separate approaches: biotintagged Ub/UbLs to modify and capture target proteins in vivo, and BioID, a tool to facilitate the labeling and identification of proximal interactors of Ub/UbL enzymes. Coupled with ongoing advances in proteomics to increase sensitivity in peptide identification, and gene-editing techniques to avoid overexpression artifacts, biotinbased systems promise to reveal new information about the role of Ub/UbL modifications in development and disease.