A technique using a fully automated on-line solid phase extraction (SPE) system (Symbiosis, Spark Holland) combined with liquid chromatography (LC)–mass spectrometry (MS/MS) has been investigated for fast bioanalytical method development, method validation and sample analysis using both conventional C 18 and monolithic columns. Online SPE LC–MS/MS methods were developed in the automated mode for the quantification of model compounds (propranolol and diclofenac) directly in rat plasma. Accuracy and precision using online SPE LC–MS/MS with conventional C 18 and monolithic columns were in the range of 88–111% and 0.5–14%, respectively. Total analysis cycle time of 4min per sample was demonstrated using the C 18 column. Monolithic column allowed for 2min total cycle time without compromising the quality and validation criteria of the method. Direct plasma sample injection without on-line SPE resulted in poor accuracy and precision in the range of 41–108% and 3–81%. Furthermore, the increase in back pressure resulted in column damage after the injection of only 60 samples.