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A novel microfluidic chromatography device coupled with tandem mass spectrometry (LC–MS/MS) was utilized for the multiplex analysis of 5 steroids (testosterone, dihydrotestosterone, progesterone, cortisol, cortisone) in human serum. The use of microfluidics allowed for reduction of the chromatographic flow rate to 3 μl/min with overall method run times comparable to standard flow LC–MS/MS methods reported in the literature, corresponding to a 150 fold decrease in solvent consumption. Furthermore, a simple sample preparation protocol was employed requiring injection of only 0.5 μl of sample, corresponding to a 100–400 fold increase in on-column sensitivity as compared to published standard flow assays. The measured LOQ for both testosterone and progesterone was 0.4 ng/mL, representing an improvement over reported literature values obtained by standard flow methods employing comparable sample …