We have developed a simple and sensitive method for the simultaneous determination of stress-related hormones such as cortisol (CRT) and dehydroepiandrosterone (DHEA) in saliva by automated online in-tube solid-phase microextraction (SPME) coupled with liquid chromatography-tandem mass spectrometry (LC-MS/MS). CRT and DHEA were separated within 5 min on a Discovery HS F5 column, with 0.2% formic acid/acetonitrile (65/35, v/v) as the mobile phase at a flow rate of 0.2 mL min(-1). Electrospray ionization conditions in the positive ion mode were optimized for MS/MS detection of CRT and DHEA. The optimum in-tube SPME conditions were 25 draw/eject cycles of 40 mL of sample at a flow rate of 200 mu L min(-1) using a Supel-Q PLOT capillary column as an extraction device. The extracted compounds were easily desorbed from the capillary by passage of the mobile phase, and no carryover was observed. Using the in-tube SPME LC-MS/MS method, we obtained good linearity of the calibration curve (r >= 0.9990) for CRT (0.005-1.0 ng mL(-1)) and DHEA (0.05-10.0 ng mL(-1)) using a stable isotope-labeled internal standard. The limits of detection (S/N = 3) for CRT and DHEA were 0.9 and 13 pg mL(-1), respectively. This method had sensitivity more than 30-fold higher than that of the direct injection method. The intra-day and inter-day precisions (relative standard deviations) were below 4.3% and 9.6% (n = 5), respectively. This method was successfully utilized to analyze CRT and DHEA in saliva samples without any other pretreatment or interference peaks, and the recoveries of CRT and DHEA spiked into saliva samples were each above 94%. This method was used to analyze changes in salivary CRT and DHEA concentrations in response to stress.