Objective: To assess the levels of adenosine deaminase (ADA) in serum in patients with sputum smear negative pulmonary tuberculosis (SNPTB) and to compare it with serum ADA levels in patients with non-tuberculous pulmonary disease - chronic obstructive pulmonary disease (COPD) and with healthy control group and to explore its validity as a diagnostic marker in serum in SNPTB patients.Methods:Three groups of study populations were made. Group I: SNPTB - 142 cases, Group II:non-tubercular pulmonary disease - COPD - 40 cases, Group III: healthy controls - 80 cases. Serum samples were collected and ADA assay was done by the method of Guisti and Galanti. Results: ADA levels (Mean±SD, U/L) in the three groups were as follows: Group I: 42.26±21.22, Group II: 23.31±8.22, Group III: 18.88±6.67. Difference between Group I and Group III was statistically significant (P < 0.0001). The test showed a high specificity 91.25% (95% confidence interval - CI 83.00 - 95.7) and a sensitivity of 83.10% (95% CI 76.08-88.37) in Group I. Positive predictive value, negative predictive value, positive likelihood ratio, negative likelihood ratio and accuracy in Group I were 94.00%, 69.52%, 9.49, 0.18 and 82.43% respectively.Conclusions: Overall assessment of the use of serum ADA levels as a diagnostic biochemical marker in smear-negative pulmonary tuberculosis patients showed promising results. Studies with a larger population group are required to validate its use as a routine diagnostic test in these cases.