Native peptides and proteins are of increasing interest in biomedical research because they hold promise to represent a large number of useful diagnostic and therapeutic biomarkers. Discovery attempts from patient samples have to deal with the complexity of biology from a disease perspective as well as with a high individual variability. High throughput screening of samples is therefore the strategy of choice to detect relevant peptidic biomarkers, and requires a high order of automation particularly in the detection process. In this contribution, a novel technical approach employing a fully automated MALDI-TOF/TOF mass spectrometer is described. This approach combines high throughput biomarker discovery with the identification of corresponding endogenous peptides in one instrument and from the same set of samples. The degree of automation allows the analysis of thousands of chromatographic fractions corresponding to up to one hundred patient samples per day. The applied relative quantification via Differential Peptide Display((R)) is performed in a label-free way and shows a dynamic range of up to four orders of magnitude in the accessible peptide concentrations. The typical limit of detection is in the mid- to low-picomolar range for body fluids such as blood plasma, urine and cerebrospinal fluid. Sequence assignment via MALDI-TOF/TOF mass spectrometry is carried out either in an overview approach, characterizing rapidly the peptide composition e.g. of a novel sample, or in a directed approach, analyzing a list of biomarker candidates deduced from statistically significant abundance differences from the biomarker discovery process.