We here report the first comparative proteomics of purified yeast post‐Golgi vesicles (PGVs). Vesicle samples isolated from PGV‐accumulating sec6‐4 mutants were treated with isobaric tags (iTRAQ) for subsequent quantitative tandem mass spectrometric analysis of protein content. After background subtraction, a total of 66 vesicle‐associated proteins were identified, including known or assumed vesicle residents as well as a fraction not previously known to be PGV associated. Vesicles isolated from cells lacking the polarity protein Sro7p contained essentially the same catalogue of proteins but showed a reduced content of a subset of cargo proteins, in agreement with a previously shown selective role for Sro7p in cargo sorting.