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Summary: To date, phase separation studies have largely been limited to in vitro assays using non-native conditions and aggregation-prone recombinant proteins that are often difficult to purify. This protocol describes the determination of relative protein concentration thresholds for phase separation through fluorescent imaging of GFP-tagged proteins in cells. The commercial availability of various plasmids and antibodies, as well as advances in gene editing, allow this procedure to be modified for the study of various phase-separating proteins in their relevant contexts.For complete details on the use and execution of this protocol, please refer to Lee et al. (2020).