Abstract Autophagy and mitophagy play a central role in cellular homeostasis. In pathological conditions, the flow of autophagy and mitophagy can be affected at multiple and distinct steps of the pathways. Unfortunately, the level of detail of current state of the art analyses does not allow detection or dissection of pathway intermediates. Moreover, is conducted in low-throughput manner on bulk cell populations. Defining autophagy and mitophagy pathway intermediates in a high-throughput manner is technologically challenging, and has not been addressed so far. Here, we overcome those limitations and developed a novel high-throughput phenotyping platform with automated high-content image analysis to assess autophagy and mitophagy pathway intermediates.