High performance liquid chromatography coupled with electrospray mass spectrometry is widely used for quantitative determination of immunosuppressive drugs (sirolimus, tacrolimus, everolimus, CsA and MPA) in biological fluids. The growth in volume for testing these drugs and economic constraints in clinical laboratories has led to heightened demand for high throughput methods. Fast-flow on-line extraction with switching valve technique and implementation of automation accelerates sample preparation. For on-line purification the combination of fast flow of washing solution and narrow-bore extraction column provides a clean sample in a very short time without compromising precision and accuracy. The unique feature of multireactant monitoring tandem mass spectrometry reduces significantly the need for chromatographic separation, as long as matrix effects are not detected, and permits simultaneous measurement of several drugs in one run when they are present in the same specimen. Additionally, the same method together with the identical sample preparation and HPLC-MS conditions and setting can be used for measurement of all five immunosuppressants, four of them in blood, MPA in plasma. Thanks to the high sensitivity of LC-MS only a small volume of biological sample is required for analysis. However for MPA quantitation, mass interference attributable to in-source fragmentation of its glucuronide metabolite can be a problem if the latter is not chromatographically separated from the parent drug before introduction of the sample into the ion source. Thus, chromatographic separation is extremely important for MPA analysis. In conclusion, important features of LC-MS methodology for immunosuppressive drugs include: shortened analysis time, increased throughput, selectivity and lower cost of analysis.<br /> <br />