Antibiotic sensitivity should be tested. In the sensitivity test, there are technical factors that influence the formation of the inhibition zone diameter. Based on several research one of the technical factors that affect the diameter of the inhibition in the disc diffusion method is the temperature incubation of the media, this must be examined so that it can be controlled to ensure the validity of the sensitivity test results. This study aims to determine the mean, difference, and analyze the diameter of the inhibition zone of the antibiotics namely Clindamycin, Levofloxacin, Tetracycline, and Trimethoprim against Staphylococcus aureus on Mueller-Hinton agar media with incubation temperatures of 33°C, 34°C, 35°C, 36°C and 37°C for 18 hours. This research is observational, with a cross-sectional design. The data used are primary data with 100 data on the diameter of the antibiotic inhibition zone, obtained by measuring the diameter of the inhibition zone with different incubation temperatures. The selection of antibiotics is based on the mechanism of action of antibiotics inhibiting bacteria namely, the cell wall or membranes that surrounds the bacterial cell; the machineries that make the nucleic acids DNA and RNA and the machinery that produce proteins (the ribosome and associated proteins) with a range of inhibition zones based on Internal Quality Control CLSI. The data will be processed univariately and the Repeated Measure statistical test to determine the significance of the difference in the diameter of the formed inhibition zone using the ANOVA test. The results of the measurement of the inhibition zone diameter on the incubation temperature variation showed a significant difference with p-value 0.000 for Levofloxacin, Tetracycline and Trimethoprim, while for p-value Clindamycin is 0.010. Levofloxacin, Tetracycline, and Trimethoprim antibiotics, the higher the incubation temperature, the average diameter of the inhibition zone is smaller, while for Clindamycin the higher the incubation temperature, the higher the average diameter of the inhibition zone is the same. There is an effect of incubation temperature volume on the diameter of the antibiotic inhibition zone in the disc diffusion method antibiotic sensitivity test. The research indicates that incubation temperature affects the diameter of the antibiotic inhibition zone in disc diffusion tests, underscoring the need for standardized and precise testing conditions to ensure accurate and reliable antibiotic sensitivity results.