The development of hypoxic areas often takes place in solid tumors and leads cells to undergo adaptive signalization like autophagy. This process is responsible for misfolded or aggregated proteins and nonfunctional organelle recycling, allowing cells to maintain their energetic status. However, it could constitute a double-edged pathway leading to both survival and cell death. So, in response to stress such as hypoxia, autophagic and apoptotic cells are often mixed. To specifically study and characterize autophagic cells and the process, we needed to develop a method able to (1) isolate autophagic subpopulation and (2) respect apoptotic and autophagic status. Sedimentation field-flow fractionation (SdFFF) was first used to monitor physical parameter changes due to the hypoxia mimetic CoCl(2) in the p53 mutated SKNBE2(c) human neuroblastoma cell line. Second, we showed that "hyperlayer" elution is able to prepare autophagic enriched populations, fraction (F3), overexpressing autophagic markers (i.e., LC3-II accumulation and punctiform organization of autophagosomes as well as cathepsin B overactivity). Conversely, the first eluted fraction exhibited apoptotic markers (caspase-3 activity and Bax increased expression). For the first time, SdFFF was employed as an analytical tool in order to discriminate apoptotic and autophagic cells, thus providing an enriched autophagic fraction consecutively to a hypoxic stress.