We describe here the effect of UV-B irradiation on the proteome and flavonoid content of the barley seedling leaf epidermis and mesophyll. Flavonoid analysis was performed using UPLC-PDA/-MS. The major flavonoid molecule responding to UV-B radiation was saponarin, and this accumulated in the epidermis, but not in the mesophyll. Changes in protein expression were determined using two-dimensional gel electrophoresis (2-DE) and identified 11 responsive proteins (seven up-regulated and two down-regulated in the epidermis; and one up- and one down-regulated in the mesophyll). A label-free LC-MS/MS(E) approach was applied for a subset of samples consisting of epidermis tissue and was able to detect a further 15 (11 up-regulated and four down-regulated) proteins. Most of the proteins with changed expression pattern after UV-B treatment were involved in initial responses characteristic for oxidative stress. Others were primary metabolism proteins involved in the supply of precursors for secondary metabolites. A separate analysis of epidermis and mesophyll tissue is important to give a spatially resolved picture of the response to UV-B treatment. The label-free LC-MS/MS(E) approach is complementary to the more conventional two-dimensional gel electrophoresis, as there was no overlap between the spectra of proteins identified by the two techniques.