In an effort to determine whether there is a major difference in protein level between isoniazid resistant and susceptible Mycobacterium tuberculosis strains, the proteins extracted from nine isoniazid (INH) monoresistant M. tuberculosis strains, seven isoniazid susceptible M. tuberculosis strains, and M. tuberculosis H37Rv, were analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). In all protein spot differences, five protein spots that are upregulated in INH-resistant strains were identified as Rv1446c, Rv3028c, Rv0491, Rv2971, and Rv2145 by matrix-assisted laser desorption/ionization-time of fight-mass spectrometry (MALDI-TOF-MS). Most of these differential proteins are membrane proteins, which are known to be rich in potential diagnostic and therapeutic protein targets. These results suggest that the differentially expressed proteins from INH-resistant strains might be used as potential immunodiagnosis antigens and candidate novel drug targets against drug-resistant tuberculosis.