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An everolimus DBS method for the oncology setting was developed and validated.The method was validated according to the EMA guidelines for bioanalytical methods.Everolimus accuracy and precision were satisfactory at hematocrit values of 0.25–0.50 L/L.A clinical validation using everolimus DBS in cancer patients is being undertaken.While the therapeutic drug monitoring (TDM) of everolimus has been routinely performed for over 10 years in solid organ transplantation medicine, in order to optimize the balance between effectiveness and toxicity, it is yet uncommon in the treatment of malignancies. The aim of this study was to develop and validate a bioanalytical method to quantify everolimus in dried blood spots (DBS) to facilitate TDM for the oncology outpatient setting. The hematocrit effect of everolimus was investigated. An 7.5 mm disk from the central part of the DBS was punched, followed by the extraction of everolimus from the DBS by methanol/acetonitrile (80/20%) spiked with deuterium-labelled everolimus as internal standard. Subsequently, everolimus was separated and analyzed using ultra performance liquid chromatography-tandem mass spectrometry (UPLC–MS/MS). The UPLC–MS/MS method was validated according to the European Medicine Agency (EMA) guideline. Everolimus concentrations could be quantified over the range of 3–75 μg/L. The intra- and inter-assay precision and accuracy of the method were shown to be acceptable (coefficient of variation ≤10.7% and relative error ≤4.4%, respectively). The matrix effects appeared to be influenced by the hematocrit effect. The hematocrit effect was tested in a range of 0.20–0.50 L/L, at which hematocrit accuracy and precision were satisfactory at values ≥0.25 L/L. However, at 0.20 L/L hematocrit in combination with high everolimus concentrations of 20 and 40 μg/L, the precision was adequate (≤7.4%), but the accuracy was >15% of the nominal concentration. Everolimus was stable in DBS for at least 80 days at 2–8 °C. Given these results, the everolimus DBS method has been successfully developed and validated. Special attention is necessary for cancer patients with both a 0.20 L/L hematocrit in combination with everolimus concentrations ≥20 μg/L. A clinical validation for the use of everolimus DBS in cancer patients is currently being undertaken.