Ifosfamide blood concentrations are necessary to monitor its therapeutic response, avoiding any adverse effect. We developed and validated an analytical method by UPLC-MS/MS to quantify ifosfamide in dried blood spots (DBS). Blood samples were collected on Whatman 903® filter paper cards. Five 3 mm disks were punched out from each dried blood spot. Acetonitrile and ethyl acetate were used for drug extraction. Chromatographic separation was carried out in an Acquity UPLC equipment with a BEH-C18 column, 2.1 x 100 mm, 1.7 μm (Waters®). The mobile phase consisted in 5 mM ammonium formate and methanol:acetonitrile (40:48:12 v/v/v) at 0.2 mL/min. LC-MS/MS detection was done by ESI+ and multiple reaction mode monitoring, ionic transitions were m/z1+ 260.99 > 91.63 for ifosfamide and 261.00 > 139.90 for cyclophosphamide (internal standard). This method was linear within a 100–10000 ng/mL range and it was accurate, precise and selective. Ifosfamide samples in DBS were stable for up to 52 days at -80°C. The procedure was tested in 14 patients, ages 1 month to 17 years (9 males and 5 females), with embryonic tumours treated with ifosfamide, alone or combined, at a public tertiary referral hospital. Ifosfamide blood levels ranged from 11.1 to 39.7 μmol/L at 12 hours after the last infusion, while 24-hour levels ranged from 0.7–19.7 μmol/L. The median at 12 hours was 19.5 μmol/L (Q25 14.4–Q75 29.0) and 3.8 μmol/L (Q25 1.5–Q75 9.9) at 24 hours, p<0.001. This method is feasible to determine ifosfamide plasma levels in paediatric patients.