During active tuberculosis a spectrum of physiologically different Mycobacterium tuberculosis bacilli reside in human tissues. Subpopulations of the pathogen survive antibiotic treatment for a prolonged time in a dormant state of phenotypic drug resistance, a phenomenon independent of genetic mutations. Here, we used an established culture model of nutrient deprivation to shift down M. tuberculosis from growth to nonreplicating survival, which is characterized by a drastic loss of drug susceptibility. Liquid chromatography coupled with mass spectrometry techniques were employed to quantify drug penetration in replicating and nutrient-starved nonreplicating bacilli. We found that intracellular concentrations of fluoroquinolones, rifamycins, and linezolid were lower in nonreplicating M. tuberculosis. Studies with pump inhibitors suggest that the observed differences were independent of efflux processes. We conclude that decreased drug permeability contributes to phenotypic drug resistance of dormant M. tuberculosis.