► Online SPE–HPLC–MS/MS method for three antitumor drugs in a combined anticancer therapy. ► Harmonized sample treatment for plasma and whole blood samples. ► Analytical method validated. ► Real samples of plasma and blood from patients under oncologic therapy were measured and results are included. Docetaxel and temsirolimus are some of the most used drugs in a wide range of solid tumors. In preclinical studies, mTOR inhibitors such as temsirolimus have demonstrated synergistic cytotoxic effects with taxanes providing the rationale for combination studies. These anticancer agents exhibit a narrow therapeutic concentration range and due to their high inter- and intra-individual pharmacokinetic variability, therapeutic dose monitoring by highly sensitive methods as LC–MS/MS are important for clinical research. Therefore, the aim of this study was to develop and validate a sensitive, fast and convenient method for the simultaneous identification and quantification of docetaxel, temsirolimus and its main metabolite, sirolimus, using paclitaxel, another anticancer drug, as the internal standard. These analytes were quantified by an integrated online solid phase extraction–high performance liquid chromatography–tandem mass spectrometry (SPE–HPLC–MS/MS) system. Separation was performed on a Zorbax eclipse XDB-C8 (150mm×4.6mm, 5μm) column. The mass spectrometer tandem quadruple detector was equipped with jet stream electrospray ionization, monitored in multiple reactions monitoring (MRM) and operated in positive mode. A combination of protein precipitation with methanol/zinc sulphate (70:30) (v/v) and online SPE using a Zorbax eclipse plus C8 (12.5mm×4.6mm, 5μm) cartridge was used to extract the compounds. This method allows the use of the same reagents, sample treatment and analytical technique independently of whether the samples are whole blood or plasma. The method has been successfully validated and applied to real samples. It is a suitable method for dose adjustment and for evaluating potential drug interactions during combined treatments.