Liquid chromatography with electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) technique is gaining more and more attraction as the method of choice for multi-sample analysis. However, it is strongly susceptible to the influence of matrix components. Matrix effects are the main source of substantial losses in detection sensitivity and have to be compensated via complex quantification methods In this work, we introduce a sophisticated quantification method for the LC-ESI-MS/MS analysis of 16 substances in urine samples using a single continuously post-column infused internal standard (PCI-IS) for matrix effect correction. The performance of the introduced technique was proven by the simultaneous quantification using internal standards. Our results demonstrate that a single post-column infused internal standard suffices to analyze multiple target analytes. The introduced method is a new approach to analyze complex matrices and represents a powerful alternative to the classic internal standard methodology. The proposed technique significantly reduces the required steps for sample preparation, costs of additional stable isotopically-labeled internal standards, and self-induced matrix effects.