Screening for latent tuberculosis infection (LTBI) prior to the prescribing of anti-TNF agents and monitoring for infection during treatment are recommended. The feasibility of novel screening tools, including QuantiFERON-TB Gold In-Tube (QFT-GIT), remains unclear in the setting of immunosuppression. The aim of this study was to evaluate the usefulness of serial QFT-GIT during biologic therapy to assess whether dynamic changes in IFN-γ levels may be helpful in identifying reactivation of LTBI or newly acquired TB. We conducted a prospective study on patient candidates to TNF inhibitors. QFT-GIT was performed at baseline and after 3 and 6 months since biologic onset. A further follow-up period of 6 months was observed. Among patients enrolled (n = 119; F = 69 %; median age = 47 years, range 18–80), 24 had at least 1 risk factor for LTBI. Ninety-six were taking immunosuppressants at the time of TB testing. At baseline, 5 patients displayed positive, 93 negative, and 21 indeterminate QFT-GIT results. We observed QFT-GIT conversions and reversions in 12 patients with LTBI and in 73 without LTBI. QFT-GIT results changed of 28 % at month 3 and of 21 % at month 6; the greatest change was observed in patients with indeterminate results that became negative (15 %; p < 0.02). No TB cases were detected. In conclusion, the routine use of both QFT-GIT and TST at screening seems not to give any advantage in the setting of patients awaiting biologics. In addition, the feasibility of serial QFT-GIT during biologic therapy needs definition since changes in IFN-γ levels may occur without a pathologic connotation.