The QuantiFERON-TB Gold In-Tube (QFT-G IT) test (Cellestis Inc., Valencia, CA) is one of the gamma interferon release assays (IGRAs) that are promising tools for diagnosing active or latent Mycobacterium tuberculosis infections. We investigated the clinical and laboratory factors that affect the rate of indeterminate QFT-G IT test results. We also suggest a workflow strategy for achieving optimized test results using the QFT-G IT test for the diagnosis of active tuberculosis (TB) or latent TB infection. We performed statistical analysis using data from a retrospective review of medical records. The first phase included 683 QFT-G IT test results from 676 patients tested between January 2008 and May 2008, and the second phase included an additional 663 QFT-G IT test results from 653 patients tested between January 2008 and December 2008 at Samsung Medical Center, a tertiary care hospital in South Korea. Immunosuppressive drug therapy, underlying diseases, bedridden status, and hypoalbuminemia were significantly associated with indeterminate QFT-G IT test results. With reduction of the incubation delay during the test procedure from an average of 9.82 h to an average of 2.70 h with changes in the workflow, the frequency of indeterminate QFT-G IT test results was significantly reduced from 11.4% to 2.7%. With<6 h of incubation delay, however, the frequency of indeterminate QFT-G IT test results was increased in a statistically significant manner. This study demonstrates that not only clinicopathological factors but also laboratory factors, such as incubation delay, significantly affect the rate of indeterminate QFT-G IT test results; therefore, optimization of the test procedure may contribute to reductions in the rate of indeterminate QFT-G IT test results, which delay the diagnosis of TB.