Updated project metadata. We thoroughly characterize isobaric labeling (TMT)-associated reporter ion interference at the MS2 level using a defined two-proteome experimental system with known ground truth. We discover remarkably poor agreement between the apparent precursor purity in the isolation window and the actual level of observed reporter ion interference in MS2-scans - a discrepancy that we find resolved by considering co-fragmentation of peptide ions hidden within the spectral “noise” of the MS1 isolation window. On this basis, we establish a comprehensive regression modeling strategy for predicting accurate, feature-wise estimates of reporter ion interference. Finally, we demonstrate the utility of accurate ion purity estimates for accurate fold change estimation and unbiased PTM site-to-protein normalization.