Skimmed milk as a protein substrate has been used for the detection of proteolytic activity. A simple and rapid method for detecting proteolytic activity is radial diffusion method. Microbial proteases are the most important for industry, and constitute of approximately 60 % of the total industrial enzyme market. There are reports on the effort to obtain new isolates that are good protease producers, and to search new species with particular characteristics that can vary with the growth substrate. This study was carried out to detect the skimmed milk-degrading ability of Rhizopus azygosporus UICC 539 on 1 % and 2 % skimmed milk at various temperatures. Agar block (diameter 6 mm) containing a concentration of R. azygosporus cells at approximately 106 cell/mL was prepared from a 5-day old fungus in Potato Sucrose Agar (PSA) at 30 °C. The agar block was inoculated on modified Czapek's Dox Agar (CDA) plates with the absence of a carbon source and by the addition of 20 % Victoria Blue as an indicator. Commercial skimmed milk at a concentration of 1 % (w/v) or 2 % (w/v) was applied as a carbon source. The CDA plates were incubated for 3 days and 5 days at various temperatures (30 °C, 35 °C, 40 °C, 45 °C and 50 °C). Modified CDA plates without the fungus served as control. Clear zones were indicative of skimmed milk hydrolysis. Enzymatic Index (EI) was calculated according to the following formula: R/r, where R was the diameter of the entire clear zone, and r was the diameter of the fungal colony. The results showed that skimmed milk-degrading ability of R. azygosporus UICC 539 was detected at both concentrations and at all tested temperatures. High EI was observed at a concentration of 1 % skimmed milk at 45 °C and 2 % skimmed milk at 50 °C. The highest EI was observed at 50 °C at 2 % skimmed milk after day-5 of incubation. In conclusion R. azygosporus UICC 539 was able to degrade skimmed milk at concentrations of 1 % and 2 %, in the temperature range of 30-50 °C and produced clear zones, which was indicative that this strain secreted proteolytic enzyme (protease) into the medium. This study indicated that R. azygosporus UICC 539 was potential to be explored for hydrolysis of protein-containing agricultural byproducts at high temperatures.