A high throughput targeted UPLC-MS/MS single platform, employing a reversed-phase gradient separation, has been developed for the quantification/monitoring of small molecule metabolites, lipids and peptides. The platform employs a single LC column and mobile phase combination which allows the analysis of multiple analyte classes with either positive or negative ion MRM detection. The use of metabolic profiling (metabonomics/metabolomics) to discover biomarkers of organismal response to environmental and physiological change is now widespread. In biomedical applications metabolic profiling is being deployed as a method for finding novel, mechanistic, biomarkers of disease with obvious potential for improving diagnosis, and patient stratification. Hypothesis driven metabolomics delivers detailed qualitative and quantitative analysis on specific pathways or classes of metabolites, allowing researchers to analyse the effects of disease or treatments in greater detail. These targeted assays usually employ “bespoke” methods which are optimized for each pathway or metabolic class making multiplexing assays difficult. We have developed a single analytical LC-MS/MS platform which is rapid, simple and reliable. The methodology employs a single LC column / mobile-phase combination which facilitate the bile acids, biogenic amine, free fatty acids, acyl carnitines, lipids and 100 protein panel. This single platform approach has been employed for the analysis of plasma from a lung and bladder cancer study, showing excellent throughput and sensitivity.