HPLC coupled to atmospheric pressure ionisation mass spectrometry has almost replaced HPLC assays with UV, fluorescence, or electrochemical detection, due to its enhanced speed, sensitivity, and selectivity, especially when tandem-MS is used. To increase the speed and sensitivity of the drug assays further, high-speed HPLC, multi-component analysis, and μHPLC are used on a routine basis. Sample preparation is recognized as an important issue in bioanalytics. The use of a 96-well plate format with automated liquid-handling systems, off-line and on-line solid-phase extraction or automated liquid-liquid extraction allows to cope with the high sample throughput enabled by LC-MS. Although LC-MS/MS represents the highest standard with respect to sensitivity and selectivity, LC-MS, as a less expensive alternative, is useful in many stages of drug discovery and development. CE-MS and CEC-MS appear to be an attractive alternative to HPLC-MS with respect to separation power, but both are a challenge in application and only seldomly used for the quantification of new drug candidates in biological fluids.