The feasibility and advantages of using sophisticated chemometric tools in combination with the execution of thoroughly planned experiments to determine experimental conditions for optimal performance of an LC-ESI-MS/MS analysis is demonstrated. A stepwise strategy is proposed, which provides a controlled optimization procedure of the chromatographic quality (in terms of separation among the sample constituents) and maximizes the mass spectrometric signal of the selected product ions. Design of experiments (DOE) and response surface methodology are applied throughout the procedure. The stepwise approach has the advantage of dealing with the different optimization criteria separately, i.e. first ensuring sufficient chromatographic separation, then maximizing the amount of precursor ion entering the mass spectrometer, and finally generating high amounts of selected product ions. The experiments are performed on a linear ion trap mass spectrometer. Retention mapping using the band-tracking model is applied during LC development, which facilitates the optimization of segmented gradients. A set of different siderophores, strong iron chelates, is used as the model substances.