Proximity dependent biotinylation is an important method to study protein-protein interactions in cells, for which an expanding number of applications has been proposed. The laborious and time consuming sample processing has limited project sizes so far. Here, we introduce an automated workflow on a liquid handler to process up to 96 samples at a time. A sample preparation workflow for enrichment and digestion of biotinylated proteins (Bartolome, A et al. bioRxiv 2022) was adapted and implemented on a liquid handler (Agilent Bravo). Here, streptavidin beads are acetylated prior to loading of the samples to decreases streptavidin contamination after on-bead digestion with LysC. In a second elution, a mixture of acetonitrile and trifluoroacetic acid is used to retrieve biotinylated peptides. As this step is time sensitive, automatization on a liquid handler improves reproducibility. In addition, the impact of the sample input as well as the LC gradient was analysed. The sample input could be reduced from 20 Mio to 8 Mio cells and the gradient shortened from 120 minutes to 21 minutes without compromising the efficiency of biotinylated protein enrichment. Taken together we were able to implement a new workflow that improves reproducibility and speeds up sample processing and measuring time for proximity labelling experiments considerably, and we successfully applied this workflow to optimize the detection of proteasome substrates by proximity-dependent labeling.