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Objectives The validation and clinical application of an LC-MS/MS method for the quantification of nevirapine in dried blood spots (DBS) and dried breast-milk spots (DBMS) are presented. Methods DBS and DBMS were prepared from 50 and 30 μL of nevirapine-spiked whole blood and human breast milk, respectively. Chromatographic separation was achieved on a reverse-phase C18 column with 0.1% formic acid in water/acetonitrile using a solvent gradient programme at a flow rate of 400 μL/min, and detection was by a TSQ Quantum Access triple quadrupole mass spectrometer. The clinical application was evaluated in HIV-positive nursing mothers and their breastfed infants. Results The assay was validated over the concentration range 50-10,000 ng/mL. Accuracy ranged from 93.3% to 113.4% and precision ranged from 1.9% to 12.0%. The mean (percentage coefficient of variation) recovery of nevirapine from DBS and DBMS was ≥ 70.7% (≤ 8.2) and the matrix effect was ≤ 1.04 (≤ 6.1). Nevirapine was stable in DBS and DBMS for ≥ 15 months at room temperature and -80°C. Mean (SD) AUC0-12, Cmax and Cmin in maternal plasma versus breast milk were 57,808 ng · h/mL (24,315) versus 55,817 ng · h/mL (22,368), 6140 ng/mL (2605) versus 5231 ng/mL (2215) and 4334 ng/mL (1880) versus 4342 ng/mL (2245), respectively. The milk-to-plasma concentration ratio over the dosing interval was 0.94 (0.15). Infant plasma concentrations 2 and 8 h after maternal dosing were 580.6 ng/mL (464.7-1607) and 584.1 ng/mL (381.5-1570), respectively. Conclusions These methods further extend opportunities for conducting clinical pharmacokinetic studies in nursing mother-infant pairs, especially in resource-limited settings.