Understanding in vivo protein–protein interactions is critical to dissect precise functions of the regulatory proteins of fungal secondary metabolites. As many fungi differentially produce a diverse array of secondary metabolites during their lifecycle, it is important to understand the cell-type specific regulation of secondary metabolism. However, due to the difficulty of sample preparation of biologically active proteins in fungal spores, protein–protein interaction studies have been generally restricted. While some outstanding studies revealed protein–protein interactions of selected regulators, including the velvet proteins in vegetative cells, a detailed protocol for investigating the protein–protein interactions in the fungal spores has not yet been reported. Here, we describe a working protocol for the purification and identification of interacting protein partners of the spores of Aspergillus nidulans employing the VelB protein as an example.