The diagnosis of latent tuberculosis (TB) infection (LTBI) is critical to improve TB treatment and control, and the T-SPOT.TB test is a commercial enzyme-linked immunosorbent spot assay used for this purpose. The objective of the study was to increase automation and extend the time between blood collection and processing for the T-SPOT.TB test from 0 to 8 h to 0 to 54 h. The previous maximum time between blood collection and processing for the T-SPOT.TB test is 32 h using T-Cell Xtend. For this, we compared the T-SPOT.TB test using manual peripheral blood mononuclear cell (PBMC) isolation by density gradient separation at 0 to 8 h (reference method, control arm) to an automated PBMC isolation method using magnetic beads (T-Cell Select kit) at 0 to 55 h postcollection. A total of 620 subjects were enrolled from 4 study sites, and blood samples were collected from each volunteer, comprising 1,850 paired samples in total. Overall agreement between both methods was 96.8% (confidence interval CI, 95.9 to 97.6%), with 95.8% (CI, 93.5 to 97.5%) positive and 97.1% negative agreement (CI, 96.1 to 97.9%). In summary, there was a strong overall agreement between the automated and manual T-SPOT.TB test processing methods. The results suggest that the T-SPOT.TB test can be processed using automated positive selection with magnetic beads using T-Cell Select to decrease hands-on time. Also, this cell isolation method allowed for the time between blood collection and processing to range from 0 to 55 h. Additional studies in larger and diverse patient populations including immunocompromised and pediatric patients are needed.