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Summary: We present an optimized protocol for in vivo affinity purification proteomics and biochemistry using the model organism C. elegans. We describe steps for target tagging, large-scale culture, affinity purification using a cryomill, mass spectrometry and validation of candidate binding proteins. Our approach has proven successful for identifying protein-protein interactions and signaling networks with verified functional relevance. Our protocol is also suitable for biochemical evaluation of protein-protein interactions in vivo.For complete details on the use and execution of this protocol, please refer to Crawley et al.,1 Giles et al.,2 and Desbois et al.3 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.