Cells are exquisitely compartmentalized to achieve precise spatiotemporal regulation of myriad processes and pathways. Phase separation offers one way to achieve territorial organization in the cellular context, via the creation of membrane-less organelles (MLOs). MLOs formed through phase separation are associated with numerous critical biological functions. Although hundreds of publications on related topics are produced each year, robust criteria for the determination of biologically meaningful phase separation are yet to be well established. Here we present some principles and propose a few guidelines for phase-separation studies in biology. Specifically, we provide an in-depth experiment pipeline for phase-separation studies, including mechanisms of the molecular driving forces, ways to correlate in vivo and in vitro observations, and strategies to relate the phase-separation phenomenon to biological functions. We also intend to contribute to streamlining the aforementioned diagnostic criteria by further stressing a few common caveats in the field.
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