This paper presents a study of the signal suppression and enhancement effects in assays based on HPLC-ESI-MS/MS detection. The major focus was to investigate the effect of signal suppression/enhancement of typical co-administered (concomitant) medications, i.e. naproxen and ibuprofen. The results demonstrate that the analyte and internal standard can experience signal enhancement up to a factor of ca 2.9 if the test analyte or internal standard co-elute with concomitant. Experimental results also demonstrate that the analyte and internal standard signal increased by a factor of ca 2.0 in the negative ion mode at physiological relevant levels of naproxen (100 µg/mL) and by a factor of ca 1.6 in the negative ion mode at physiological relevant level of ibuprofen (10 µg/mL) in both neat and plasma samples. Signal enhancement significantly increased when concomitant medications ionized in the same ion mode as the analyte and internal standard. To overcome signal enhancement or potential suppression from concomitant medications, a comprehensive HPLC method needs to be developed with sufficient separation of concomitant medication from the analyte and internal standard. Other means to reduce signal enhancement or potential suppression include switching ionization polarity and performing comprehensive sample clean-up to remove concomitant medications before analysis. Copyright © 2007 John Wiley & Sons, Ltd.