Here we report a strategy for rapid method development of high-throughput bioanalytical assays using ultra high-performance liquid chromatography coupled with tandem mass spectrometry (uHPLC-MS/MS). First, a data set was established for the removal of representative phospholipids under different sample treatments to guide subsequent method development for various compounds. The recovery information of the analyte(s) of interest under different extraction conditions was then obtained during method development. With the recovery profiles and the pre-established knowledge of phospholipids removal in place, an optimal extraction condition was identified to give not only a satisfactory recovery but also a good cleanup of the sample. A rapid LC or uHPLC method was developed without the need of extensive column wash after the elution of the analyte. This strategy was demonstrated through the method development of a uHPLC-MS/MS bioanalytical assay for the quantitation of ketoconazole in human plasma with liquid-liquid extraction using a hexane and ethyl acetate solvent system. The retention time for ketoconazole through an isocratic elution was 18 s. Good accuracy and precision were obtained. Assay ruggedness was demonstrated by consistent internal standard responses and retention time for 500 sequential injections. In addition, consistent results were obtained for incurred sample reanalysis.