A critical element of tuberculosis control is early and sensitive diagnosis of infection and disease. Our laboratories recently showed that different stages of disease were distinguishable via two-dimensional western blot analyses of Mycobacterium tuberculosis culture filtrate proteins. However, this methodology is not suitable for high-throughput testing. Advances in protein microarray technology provide a realistic mechanism to screen a large number of serum samples against thousands of proteins to identify biomarkers of disease states. Techniques were established for separation of native M. tuberculosis cytosol and culture filtrate proteins, resulting in 960 unique protein fractions that were used to generate protein microarrays. Evaluation of serological reactivity from 42 patients in three tuberculosis disease states and healthy PPD+ individuals demonstrated that HIV negative cavitary- and noncavitary-TB patients recognized 126 and 59 fractions, respectively. Sera from HIV patients coinfected with TB recognized 20 fractions, of which five overlapped with those recognized by non-HIV TB patients and 15 were unique to the HIV+TB+ disease state. Identification of antigens within the reactive fractions yielded eleven products recognized by both cavitary- and noncavitary-TB patients, and four proteins (HspX, MPT64, PstS1, and TrxC) specific to cavitary-TB patients. Moreover, four novel B cell antigens (BfrB, LppZ, SodC, and TrxC) of human tuberculosis were identified.