AbstractMicroproteins are a novel class of bioactive polypeptides that are encoded by small open reading frames (smORFs). Emerging evidence has demonstrated that these microproteins possess significant roles and undertake essential regulatory functions in living organisms. Meanwhile, several microproteins exhibit variable expression and distinct regulation in disease and healthy cells, revealing potential therapeutic interventions. A promising strategy to characterize microproteins and investigate their pathological association is through identification of microprotein‐protein interactions (MPIs), emphasizing the value of chemical biology tools for mapping MPIs. In this study, we applied twin‐strep tagging and TurboID proximity labeling strategies to unveil complicated cellular networks for microproteins. Encouraged by the success of both approaches in identifying the well‐studied CYREN‐Ku70/Ku80 complex, we investigated an uncharacterized FAM229B microprotein and found that it interacts with the thyroid receptor‐interacting protein 6 (TRIP6) and may regulate its nuclear translocation. Our study demonstrated that twin‐strep tagging and TurboID labeling strategies exhibited encouraging performance in identifying bona fide microprotein interacting partners.