The genera Aspergilli presents a great variety of economically important species due to their potential of industrial enzymes and harmful toxin production. In this work, a purification methodology of the major acid protease from Aspergillus clavatus was established. A suspension of seven-day-old conidia was inoculated in Vogel´s media supplemented with 1 % (w/v) glucose and 1 % (w/v) gelatin, incubated for three days, at 30 ºC  and 120 rpm. The crude extract was used as enzyme source. The acid protease activity was evaluated in 100 mM citrate buffer pH 5.0 using 2 % (w/v) hemoglobin as substrate in urea solution. In appropriate intervals, the reactions were interrupted by trichloroacetic acid. The samples were centrifuged and the absorbance was read at 280 nm. One unit of enzymatic activity was defined as the absorbance increase of 0.1, per mL, per minute. Total protein was determined by the modified Bradford´s method using bovine serum albumin as standard. Glycerol, DTT, EDTA and PMSF were assayed as protective agents against enzyme degradation. In the presence of 10 % glycerol, 1mM DTT and 2 mM EDTA the acid protease activity was preserved. The enzyme was precipitated in the range of 40 to 90 % of ammonium sulfate saturation and precipitated was dissolved. This sample was dialyzed and evaluated for its adsorption in ion exchange resins. The most part of enzyme activity linked to CM-Sephadex C-50, equilibrated in 50 mM sodium citrate buffer pH 4.0. So, a column containing the specified resin was used with flow rate of 45 mL/ h. Fractions of 3 mL were collected, analyzed and those with high enzymatic activity were pooled. The acid protease was recuperated in 37.9 % and purified 37.2-fold exhibiting in SDS-PAGE only one band with molecular weight of 30.4 kDa.