Proximity-dependent biotinylation (PDB) is a powerful means of exploring the cellular environments in which proteins reside. Expressing a protein of interest (bait) fused to a biotin ligase and adding biotin induces the covalent biotinylation of proximal partners, which are recovered on streptavidin beads and identified by MS. However, a major technical limitation of PDB is peptide carryover into subsequent MS runs. This is mitigated via lengthy inter-sample washing, considerably lowering throughput. This study aims to optimize PDB sample acquisition using an EvoSep LC system coupled to a timsTOF mass spectrometer, which has higher throughput and sensitivity than our current system, with less carryover. Our efforts resulted in an ~15-fold increase in throughput using the 60 samples-per-day (SPD) gradient with better sensitivity, identifying nearly double the proteins found by our previously standardized workflow. Significance scoring also revealed more sensitive detection of high-confidence proximal interactions (~1.5-fold) for five well-characterized baits, validating the new experimental workflow. Importantly, carryover was extremely limited, even without inter-sample washing, and limited to easily filterable abundant proteins. Without washing, this method can process 60 samples per day, using 1/16th of the sample amount previously required.