Surface Enhanced Laser/Desorption Ionization-time of flight (SELDI-TOF) mass spectrometry is a technique uniquely suited to the study of the urine proteome due to its salt tolerance, high-throughput, and small sample requirements. However, due to the extreme sensitivity of the technique, sample collection and storage conditions, as well as instrument protocols and analysis conditions, must be rigorously controlled to ensure that data generated and collected is accurate and free from artifacts. Robust and reproducible data sets can be generated and compared between clinical sites when experimental protocols are carefully standardized. This chapter aims to review known factors that cause irreproducible results so that the experiments may be designed with appropriate sample and process controls for successful biomarker discovery. A suggested protocol follows the review. A number of issues for study design are discussed and these are generally applicable to biomarker discovery experiments.