AbstractAffinity purification coupled to mass spectrometry (AP‐MS) represents a powerful and proven approach for the analysis of protein–protein interactions. However, the detection of true interactions for proteins that are commonly considered background contaminants is currently a limitation of AP‐MS. Here using spectral counts and the new statistical tool, Significance Analysis of INTeractome (SAINT), true interaction between the serine/threonine protein phosphatase 5 (PP5) and a chaperonin, heat shock protein 90 (Hsp90), is discerned. Furthermore, we report and validate a new interaction between PP5 and an Hsp90 adaptor protein, stress‐induced phosphoprotein 1 (STIP1; HOP). Mutation of PP5, replacing key basic amino acids (K97A and R101A) in the tetratricopeptide repeat (TPR) region known to be necessary for the interactions with Hsp90, abolished both the known interaction of PP5 with cell division cycle 37 homolog and the novel interaction of PP5 with stress‐induced phosphoprotein 1. Taken together, the results presented demonstrate the usefulness of label‐free quantitative proteomics and statistical tools to discriminate between noise and true interactions, even for proteins normally considered as background contaminants.