An increasing number of quantitative bioanalyses need to be performed on samples available in limited volumes, such as pharmacokinetic studies on small animals. In this context, microfluidic systems as the LC-chip device coupled to a mass spectrometer combine small sample volume requirements and high sensitivity. In this study, we present the development of a microfluidics-based method for fluoxetine (FLX) and norfluoxetine (NFL) quantitation dedicated to pharmacokinetic investigations in the rat serum. Using the methodology of experimental design, LC parameters were optimised in terms of peak resolution, analysis time, and sensitivity. An SPE method was then developed for serum samples on miniaturised 96-well plates containing a mixed-mode strong cation exchanger that provided very clean extracts with good analyte recovery (>= 66.0%). The total SPE-LC-MS/MS process required only 20 mu L per sample and the method provided a good sensitivity in a total run time of 12 min. Finally, the developed method for FLX and NFL quantitation in rat serum was fully validated. After having selected the most appropriate regression model on the basis of the accuracy profiles, method selectivity, trueness, precision, accuracy and linearity were demonstrated.