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To fully understand any cellular process, we not only need to identify the proteins implicated, but also how the protein network is structurally and spatially organized and changes over time. However, the dynamic nature of many protein interactions involved in cellular signaling pathways continues to be the bottleneck in mapping and studying protein networks. Fortunately, a recently developed proximity labeling method using engineered ascorbic acid peroxidase 2 (APEX2) in mammalian cells allows the identification of weak and/or transient protein interactions with spatial and temporal resolution. Here, we describe a protocol for successfully using the APEX2-proximity labeling method in Dictyostelium , using the cAMP receptor cAR1 as example. Coupled to the identification of the labeled proteins by mass spectrometry, this method expands Dictyostelium's proteomics toolbox and should be widely useful for identifying interacting partners involved in a variety of biological processes in Dictyostelium ., (© 2013-2023 The Journal of Biological Methods, All rights reserved.)