Raman spectroscopy is a non-destructive and label-free technique. Wavelength modulated Raman (WMR) spectroscopy was applied to investigate Mycobacterium tuberculosis cell state, lipid rich (LR) and lipid poor (LP). Compared to LP cells, LR cells can be up to 40 times more resistant to key antibiotic regimens. Using this methodology single lipid rich (LR) from lipid poor (LP) bacteria can be differentiated with both high sensitivity and specificity. It can also be used to investigate experimentally infected frozen tissue sections where both cell types can be differentiated. This methodology could be utilized to study the phenotype of mycobacterial cells in other tissues.