In comparison to the Quantiferon (QFT) test, a new principle of a method for laboratory diagnosis of latent Mycobacterium tuberculosis infection (LBTI) is provided. The new elements, as well as the calculation and introduction of an Index of Stimulation (IS), were based on measurements of tumour necrosis factor (TNF-) release stimulated by peptides (TB-P) derived from Mycobacterium tuberculosis (Mtb) related proteins and Purified Protein Derivatives in whole human blood (PPD). ELISA was used to determine the quantity of TNF-. The formula TNF(pg/ml)TB-P/ TNF-(pg/ml)PPD was used to get the IS values. The study included 15 healthcare workers who dealt with Mtb-infected patients (HCWTB), as well as 14 healthcare workers who were not exposed to Mtb patients (HCW-C). If IS was greater than 1.50, the diagnosis of LTBI was made. The number of LTBI in the HCW-TB group was 6/15 by the new technique and 9/15 by QFT. QFT produced 3/9 false-positive outcomes when tested clinically. By both procedures, all individuals of the HCW-C group were LTBI negative. There were no false positive results with this novel test. Although these are preliminary results based on a small number of patients, they are promising. However, theoretical advances in the method's principle: calculating IS from TNF—TB-P/ TNF—PPD values may indicate a step forward in the individual laboratory diagnosis of LTBI in future investigations