BACKGROUND:: Anti-T lymphocyte globulin (ATLG) modulates the alloreactivity of T lymphocytes, reducing the risk of immunological post–transplant complications, in particular rejection and graft-versus-host disease (GvHD), after allogeneic hematopoietic stem cell transplantation (HSCT).We developed and validated a new enzyme-linked immunosorbent assay (ELISA) method to measure serum levels of total ATLG and evaluate the pharmacokinetics (PK) of the drug in children with β-Thalassemia, receiving allogeneic HSCT. METHODS:: Diluted serum samples were incubated with Goat-anti-Rabbit IgG antibody coated on a microtiter plate and, then, with Goat-anti-Human IgG labelled with horseradish peroxidase. After incubation and washings, substrate solution was added and absorbance was read at 492nm. ATLG concentrations in samples were determined by interpolation from a standard curve (range: 200-0.095ng/mL), prepared by diluting a known amount of ATLG in phosphate buffered saline (PBS). Low, medium and high quality controls concentrations were 1.56, 6.25 and 25ng/mL, respectively. This method was developed and validated within the acceptance criteria in compliance with the Guidelines for a biological method validation: the sensitivity of the method was 0.095ng/mL. We analyzed serum samples from14 children with β-Thalassemia who received ATLG (Grafalon®) at a dose of 10 mg/kg administered as intravenous (IV) infusion on day-5, -4 and -3 before HSCT (day 0). Blood sampling for PK evaluation was performed on day-5, -4 and -3 before and after drug infusion; and then from day-2 to +56. RESULTS:: The median total ATLG levels pre and post IV were 0µg/mL and 118µg/mL on day -5; 85.9µg/mL and 199.2µg/mL on day -4; 153µg/mL and 270.9µg/mL on day-3, respectively.The median PK values of CL was 0.0029 (range: 0.0028-0.0057) L/kg/days, Vd was 0.088 (range: 0.025-0.448) L/kg and t1/2 was 20.2 (range: 5.8-50.2) days. CONCLUSIONS:: These data suggest that, given the marked inter-individual variability of total ATLG disposition, the development of a validated ELISA method and the possibility to measure PK parameters in paediatric populations are essential steps to optimize drug therapeutic regimens. Copyright © 2017 Wolters Kluwer Health, Inc. All rights reserved.