The present invention is directed to a method for inactivation and/or extraction of acid-fast bacteria (e.g., mycobacteria or nocardia), the method comprising the following sequential steps: (a) acquiring a test sample known to contain or that may contain acid-fast bacteria and suspending the test sample in a container containing ethanol and beads; (b) bead beating and/or vortexing the container to break up clumps and/or disrupt acid-fast bacteria cells in the container; and (c) subsequently incubating the suspension for at least about 3 minutes at room temperature to inactivate any acid-fast bacteria contained in the test sample. In accordance with the present invention, the test sample can subsequently be pelleted by centrifugation, resuspended with formic acid, acetonitrile added, and subjected to mass spectrometry for characterization and/or identification of the acid-fast bacteria.