In the present discusion, uses of experimental design for optimization of liquid chromatography tandem mass spectrometry (LC-MS/MS) integrated methods are described. A number of questions are put forward and discussed, such as: What are the optimization criteria, how do we implement appropriate optimization strategies/procedures, and how do we interpret the data obtained? Our intention has been to present and explore different parameters associated with an LC-MS/MS hyphenated experimental set-up, utilizing mainly electrospray ionization (ESI). The application of different mathematical tools is therefore a prerequisite for the realization of the robust results; Possible restrictions when it comes to choosing the setting of a specific parameter are discussed. A stepwise optimization strategy using an experimental design is also proposed, that hopefully will aid the reader to optimize the performance of such an experimental design approach.