Infection by Mycobacterium tuberculosis (Mtb) represents a significant healthcare burden, with approximately 10 million cases of human tuberculosis and 1.6 million deaths reported in 2017 by the WHO. As a result, there is an urgent need to develop new and more effective anti-tuberculosis drugs. However, the development of new antibiotics and the assessment of their toxicity represent an important challenge. Mtb is a successful intracellular pathogen that infects alveolar macrophages. Once inside, Mtb is able to resist hostile conditions such as acidic pH, osmotic stress, nitric oxide and reactive oxygen species, and it can adapt to these environmental cues. The second messenger 3',5'-cyclic adenosine monophosphate (cAMP) has been proposed to be involved in the adaptation of Mtb within the macrophage, which is rich in cholesterol and fatty acids. In this thesis, the link between osmotic stress and cAMP in mycobacteria is investigated. Thus, I provide the first evidence that in Mtb is the cAMP receptor protein (CRPMt) responsible for the production of high levels of cAMP after exposure to 250 mM NaCl. Then the role of Rv0998, a cAMP-dependent lysine acetyltransferase in Mtb physiology was investigated. Hence it was revealed that albumin is involved in the survival of an Mtb mutant lacking Rv0998 in acidic pH. Finally, I propose an alternative readout for addressing antibiotic toxicity by looking at changes in the lipidome on intact and unprocessed cells by matrix-assisted laser desorption ionisation mass spectrometry. Throughout this thesis, a combination of mass spectrometry approaches and tissue culture assays are used to understand Mtb adaptation and antibiotic toxicity. The data obtained in this thesis may help develop new strategies to combat the increasing threat that tuberculosis poses. Furthermore, it will help to rapidly assess the toxicity of aminoglycosides in HeLa and primary cells.