In recent years, different approaches have been taken to improve chromatographic performance in terms of analysis time and/or resolution. The use of columns packed with sub-2μm particles in ultra-high-pressure liquid chromatography (UHPLC) has become a technique of choice in many laboratories. Furthermore, for the analysis of complex matrices (e.g., biological fluids, plant extracts, and food and environmental samples), coupling UHPLC with mass spectrometry (MS) or tandem MS (MS2) provides a powerful analytical tool. This review describes major advances in the field of UHPLC-MS and UHPLC-MS2. We strongly emphasize the possibility of speeding up bioanalysis, drug metabolism, and multi-residue screening assays, while maintaining qualitative and quantitative performance equivalent to HPLC-MS and HPLC-MS2. We also report the possibility of gaining additional information in metabolomics, using high-resolution UHPLC with a time-of-flight analyzer. The studies summarized are discussed in this review in terms of throughput increases and resolution enhancements afforded by UHPLC. In addition, we highlight the impact of UHPLC conditions on MS detection capabilities (e.g., acquisition rate, limits of detection and matrix effects)