In an effort to create faster and more efficient bioanalytical methods for drug development, many investigators have evaluated a variety of SPE-MS systems. Over the past 15 years online systems have evolved from run times of >1.5 min/sample to <10 s/sample. High-throughput SPE-MS methods for in vitro absorption, distribution, metabolism and excretion screening assays have been described by several laboratories and shown to produce results comparable to conventional LC-MS/MS systems. While quantitative analysis of small molecules in biological matrixes holds many challenges, for several applications SPE-MS methods have achieved comparable results to LC-MS/MS with the benefit of 10-30-times the throughput. Based on its distinct advantages of throughput and streamlined workflow efficiencies, SPE-MS is a useful tool for the analysis of many in vitro absorption, distribution, metabolism and excretion assays and in vivo bioanalytical studies. Further development of SPE-MS methods and analysis workflows has the potential to expand the capabilities of this technology for other challenging bioanalytical applications.