ABSTRACT: Senescence is the last developmental phase of plant tissues, plant organs and, in the case of monocarpic senescence, entire plants. In monocarpic crops such as barley, it leads to massive remobilization of nitrogen (primarily from degradation of photosynthetic proteins) and other nutrients to developing seeds. Senescence has therefore a major impact on both yield and seed/grain quality. To further investigate this process, a proteomic comparison of flag leaves of late-senescing barley variety ‘Karl’ and a near-isogenic early-senescing line, ‘10_11’, was performed at 14 and 21 days past anthesis, using both two-dimensional gel-based and label-free quantitative mass spectrometry-based (‘shotgun’) proteomic techniques. Overall, this approach identified >9,000 barley proteins, and one-third of them were quantified. Analysis focused on proteins that were significantly (P-value ≤0.05; difference ≥1.5-fold) upregulated in early-senescing line ‘10_11’ as compared to ‘Karl’, as these may be functionally important for the senescence process. Many proteins in this group, including several membrane and intracellular receptors, glucanases, enzymes with possible roles in cuticle modification, classical pathogenesis-related proteins, membrane transporters and proteins involved in DNA repair, have likely or putative functions in plant pathogen defense. Additionally, several proteases and elements of the ubiquitin-proteasome system were upregulated in line ‘10_11’; these proteins may be involved in nitrogen remobilization, and in the regulation of both senescence and plant defense reactions. Together, our data shed new light, at the protein level, on the importance of plant defense reactions during senescence, on senescence regulation, and possibly on crosstalk between senescence regulation and plant-pathogen interaction.