Western blotting (WB) is widely used to test antibody specificity, but the assay has low throughput and precision. Here we show that an inverted version of WB allows parallel readout with antibody arrays, shotgun mass spectrometry (MS) and immunoprecipitation followed by MS (IP-MS). The pipeline provides means for large-scale implementation of concepts proposed by an international working group on antibody validation (IWGAV).