In the current project, we aimed to discover how the barley anther proteome changes, both quantitatively and qualitatively, during meiotic prophase I when the important process of homologous recombination is taking place. Taking advantage of a recently published barley anther transcriptomic dataset (BAnTr) and a newly developed sensitive mass spectrometry-based approach to analyse barley anther proteome, we have conducted high-resolution mass spectrometry analysis of barley anthers, collected at 6 time points and representing their development from pre-meiosis to metaphase. Each time point was carefully staged using immunocytology. We identified >6400 proteins, including 82 known and putative meiotic proteins, and were able to quantify dynamic variation in the proteome during different stages of barley anther development.