Rab proteins are small GTPases that are crucial for membrane trafficking. Rab1 and Rab5 are essential for cell growth and survival and are known to regulate endoplasmic reticulum (ER)-to-Golgi and early endosome trafficking, respectively. However, generating knockout systems to study their cellular roles is not feasible. Auxin-inducible degron (AID) technology is an alternative approach that can be used to establish conditional knockout (CKO) systems. Here, the protein of interest is AID-tagged and, upon recognition and ubiquitylation by an F-box ubiquitin ligase complex, is rapidly degraded by the proteasome. Now, Mitsunori Fukuda and colleagues (Hatoyama et al., 2021) use this protein degradation system to study Rab1 and Rab5. After establishing CKO cells for Rab1 and Rab5 using the AID system, the authors confirm that Rab1 is essential for the maintenance of Golgi morphology and ER-to-Golgi trafficking, whereas Rab5 is vital for early and late endosome formation, consistent with previous reports. Interestingly, early endosomes accumulate in the perinuclear region in Rab1-CKO cells, suggesting that Rab1 also participates in the spatial distribution of endocytic organelles. By contrast, experiments addressing endocytic cargo uptake in Rab5-CKO cells reveal a role in the proper maturation of endosomes through affecting receptor-mediated and fluid-phase endocytic pathways. Overall, this study presents new CKO models that help elucidate the functions of Rab1 and Rab5.