Since the sampling of tear fluid seems rather variable we sought to evaluate a protocol for combined metabolomic and proteomic analyses with regard to clinical applicability. Tear fluid was taken from twelve human volunteers at different time points using specially designed Schirmer strips. Following liquid extraction of metabolites from standardized punchesthe remaining material was processed for bottom-up proteomics. The targeted proteomic analysis of 15 tear proteins revealed a mean intra-individual variation of 23 % merely for the three most abundant proteins. Overall, our newly established workflow opens perspectives for clinical and routine diagnostic applications using well accessible tear fluid.