In this review we will give an overview of the issues related to biomarker discovery studies with a focus on liquid chromatography-mass spectrometry (LC-MS) methods. Biomarker discovery is based on a close collaboration between clinicians, analytical scientists and chemometritians/statisticians. It is critical to define the final purpose of a biomarker or biomarker pattern at the onset of the study and to select case and control samples accordingly. This is followed by designing the experiment, starting with the sampling strategy, sample collection, storage and separation protocols, choice and validation of the quantitative profiling platform followed by data processing, statistical analysis and validation workflows. Biomarker candidates that result after statistical validation should be submitted for further validation and, ideally, be connected to the disease mechanism after their identification. Since most discovery studies work with a relatively small number of samples, it is necessary to assess the specificity and sensitivity of a given biomarker-based assay in a larger set of independent samples, preferably analyzed at another clinical center. Targeted analytical methods of higher throughput than the original discovery method are needed at this point and LC-tandem mass spectrometry is gaining acceptance in this field. Throughout this review, we will focus on possible sources of variance and how they can be assessed and reduced in order to avoid false positives and to reduce the number of false negatives in biomarker discovery research.