OBJECTIVE QuantiFERON-TB Gold In-Tube (QFT-IT) assay is a highly sensitive and specific test for the diagnosis of latent tuberculosis infection. Data on the use of QFT-IT assay in children are scarce and contradictory. The aim of the study was to assess the rate of indeterminate test results and to identify factors contributing to indeterminate results on routine use of QFT-IT assay in nonimmunosuppressed children. METHODS This retrospective study included 2173 children with ages ranging from 1 month to 18 years. Determination of interferon-gamma (IFN-γ) in peripheral blood was performed by commercial QFT-IT assay. RESULTS Indeterminate test results were recorded in ten (0.46%) subjects with ages ranging from 15 months to 15 years. The value of negative control was >8.0 kIU/L in one subject, whereas in the remaining nine subjects indeterminate results were consequential to positive control (<0.50 kIU/L). None of these subjects had any history data on congenital or acquired immunodeficiency disorders. Bacterial infection (with elevated body temperature and therapy with β-lactam antibiotics) was present in eight subjects with indeterminate results, one subject had exacerbation of asthma (therapy with inhalation corticosteroids), and one subject was clinically healthy. Repeat IFN-γ determination performed in seven subjects did not yield indeterminate results. CONCLUSIONS Study results showed the rate of indeterminate QFT-IT results in nonimmunosuppressed children of all age groups to be very low. QFT-IT should preferably be performed upon resolution of acute inflammation in order to avoid repeat testing of indeterminate results in a new blood sample and to reduce the cost of testing.