Significance Half of eukaryotic proteins reside in organelles to which they are directed by dedicated targeting pathways, each recognizing unique targeting signals. Multiple proteins compete for any targeting pathway and might have different priority of reaching an organelle. However, the proteins with targeting priority, and the mechanisms underlying it, have not been explored. We developed a systematic tool to study targeting priority. We expressed a competitor protein and examined how it affects the localization of all other proteins targeted by the same pathway. We found several proteins with high targeting priority, dissected the mechanism of priority, and suggest that priority is governed by different parameters. This approach can be modified to study targeting priority in various organelles, cell types, and organisms.