Mycobacteria possess a complex and waxy cell wall comprising a large panel of glycolipids. Among these, trehalose monomycolate (TMM) represents abundant and crucial components for the elaboration of the mycomembrane. TMM is synthesized in the cytoplasmic compartment and translocated across the inner membrane by the MmpL3 transporter. Inhibitors impeding TMM transport by targeting MmpL3 show great promises as new antimycobacterials. The recent X-ray or Cryo-EM structures of MmpL3 complexed to TMM or its inhibitors have shed light on the mechanisms of TMM transport and inhibition. So far, purification procedures mainly involved the use of n-Dodecyl-ß-D-Maltopyranoside to solubilize and stabilize MmpL3 from Mycobacterium smegmatis (MmpL3_Msm) or Lauryl Maltose Neopentyl Glycol for MmpL3 from Mycobacterium tuberculosis. Herein, we explored the possibility to solubilize and stabilize MmpL3 with other detergents. We demonstrate that several surfactants from the ionic, non-ionic and zwitterionic classes are prone to solubilize MmpL3_Msm expressed in Escherichia coli. The capacity of these detergents to stabilize MmpL3_Msm was evaluated by size-exclusion chromatography and thermal stability. This study unraveled three new detergents DM, LDAO and sodium deoxycholate that favor solubilization and stabilization of MmpL3_Msm in solution. In addition, we report a protocol that allows reconstitution of MmpL3_Msm into peptidiscs.