Treatment for tuberculosis (TB) using the standard oral antibiotic regimen is effective but inefficient, requiring high drug dosing and lengthy treatment times. Three concurrent first-line antibiotics recommended by the World Health Organization (WHO) guidelines are pyrazinamide, rifampicin and isoniazid. Combining these antibiotics in a novel formulation for dry powder inhalation (DPI) may facilitate rapid and efficient resolution of local and systemic infection. However, spray-dried individually, these antibiotics were found to be physically unstable. A solution of the three antibiotics, at the WHO-recommended ratio, was spray-dried. The collected powder was assessed by a series of in vitro methods to investigate aerosol performance, particle physico-chemical characteristics and dissolution profile. Particles obtained were spherical with a surface composed primarily of rifampicin, as identified by TOF-SIMS. A mass median aerodynamic diameter of 3.5 ± 0.1 μm and fine particle fraction (