LAUSR

&NA; Miltefosine is an oral agent against the neglected tropical disease leishmaniasis, which is mostly endemic in resource‐poor areas. Dried blood spot (DBS) sampling is an attractive alternative to plasma sampling for pharmacokinetic studies in these remote areas, but introduces additional variability in analyte quantification due to possible blood spot inhomogeneity and variability in blood spot volume and haematocrit values. Volumetric absorptive microsampling (VAMS) potentially overcomes a few of these issues as the VAMS device absorbs a fixed volume that is processed as a whole. We developed and validated an LC–MS/MS method for the quantification of miltefosine with this novel sampling technique with good performance in terms of linearity, selectivity, accuracy (bias within ±10.8%), precision (CV% ≤ 11.9%), recovery, carry‐over and matrix effect. VAMS samples were stable for at least one month at room temperature and 37 °C. The impact of haematocrit on assay accuracy was reduced compared to conventional DBS sampling, but indicated a declining recovery with increased haematocrit due to haematocrit dependency in recovery from the sampling device. A clinical validation will be required to investigate whether VAMS is an appropriate and cost‐effective alternative sampling method to conventional DBS sampling. HighlightsVolumetric absorptive microsampling (VAMS) method validated for miltefosine quantification in human blood.This novel matrix sampling method eliminates variabilities due to blood spot volume and blood spot homogeneity.Developed fast and simple pre‐treatment method with reproducible recovery.Assay performed well (bias within ±10.8%, CV% ≤ 11.9%) in wide validated range of 10–5000 ng/mL.Haematocrit impact on method accuracy reduced compared to conventional DBS sampling.