LC-MS analysis of in vivo xenobiotic and endogenous components plays a key in drug metabolism and metabolomic studies, which is much more challenging than in vitro analysis due to chemical complexity, impurity interferences, limited amount of samples, low concentrations.Although various new and advanced mass spectrometers and data-processing technologies have been developed in the past decade, they are not always available in most labs.Therefore, it is very important to develop task-specific methodologies to overcome challenges by fully utilizing existing instruments and software features.Here we will present two approaches developed in our lab to meet analytical needs by using our current LC-MS capabilities.The first example was the application of a common triple quadruple instrument to quantitative analysis of human hormones in plasma.By using off-line and online derivatization, we were able to obtain targeted quantitative measurements of 20 human hormones.Compared with a reported derivation method, our method provided a wider coverage of free hormones in hormone metabolism pathways with better sensitivity.