Tuberculosis is the second most deadly infectiousdisease, surpassed only by HIV/AIDS, and hasresulted in over 1 billion deaths in the last200 years. The World Health Organization estimatesthat in 2014, 9.6 million people were infected bythis disease and 1.5 million had died. First-choicetreatment consists of fixed-dose combinationtablets containing rifampicin, isoniazid,pyrazinamide, and ethambutol hydrochloride(4-FDC). There are pharmacopeial protocolsavailable to test 4-FDC, but they are prolonged,two-step methods. One single-step method in theliterature performs the simultaneous determinationby HPLC, but requires a long acquisition time.In this context, an ultra-HPLC (UHPLC) methodwas developed based on the HPLC method withthe objective of reducing analysis time.A C18 column (1.9 μm particle size) was used withUV-diode-array detection at 238 and 282 nm. Themethod was found to be selective, linear, exact,precise, and robust. Samples from two batcheswere analyzed and the results compared with thoseobtained by the HPLC method, with no statisticallysignificant differences observed (<em>P</em> > 0.05). ThisUHPLC method reduced the analysis time from17 to 4 min, with a more than 90% reduction insample and reagent consumption and a financialeconomy of almost 50-fold.