We present an optimized protocol for in&#
xa0;vivo affinity purification proteomics and biochemistry using the model organism C.&#
xa0;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&#
xa0;vivo. For complete details on the use and execution of this protocol, please refer to Crawley et&#
xa0;al.,<sup>1</sup> Giles et&#
xa0;al.,<sup>2</sup> and Desbois et&#
xa0;al.<sup>3</sup>.