The delineation of protein–protein interactions through proximity-dependent biotinylation, particularly through the use of biotin ligases such as BioID, has established itself over time as a pivotal method in molecular biology for studying cellular processes in situ. Here, we detail a universally applicable protocol for preparing a diverse array of biological samples for BioID analysis, focusing on the steps from sample isolation to readiness for mass spectrometric analysis. Key steps outlined include mechanical lysis through sonication and freeze-thaw cycles, protein concentration normalization, and the removal of excess biotin through desalting before LC-MS processing. The protocol is designed to standardize sample preparation across diverse biological origins, encompassing a wide array of sample types such as plant tissues, bacterial cultures, and eukaryotic cells or tissues from various species. This adaptability ensures that minimal adjustments are needed for different sample types, thereby standardizing the preparation process to enhance the reliability and comparability of BioID analyses across diverse biological studies.