Introduction: S. aureus is one of the causes of death in nosocomial infection, and therefore necessary to diagnosis quickly and accurately, such as by using DNA-based Polimerase Chain Reaction (PCR). Generally, the method of DNA isolation uses a filter-based kit method, but several studies also attempt heat treatment as a simplification of the DNA isolation method. However, the quality and quantity of the heat treatment compared filter-based kit method to is unknown. This research aims to compare heat treatment with filter-based kit method based on the Limit of Detection (LoD) and the Limit of Quantification (LoQ). Research Method: This research used laboratory experimental in vitro. DNA isolation S.aureus bacteria with ATCC code 14922 uses filter based kit and heat treatment on bacterial concentration 101,102,103,104, and control. The method was evaluated by LoD and LoQ based on the calculation of DNA concentration (yield) and purity of extracted DNA (purity). Data were analyzed using Kruskal wallis and followed by post hoc analitic Mann whitney significant if p<0.05. Result & Discussion: Based on DNA yield resulted in an LoD 2 ng/µl and an LoQ 2 ng/µl. This value was detected in 101 CFU/ml concentration (yield 5±1,73 ng/µl). Meanwhile, heat-treatment resulted in an LoD of 2,06 ng/µl and an LoQ 6,11 ng/µl. This value was detected ini 104 CFU/ml bacterial concentrations (yield 3,67±0,57 ng/µl). The purity obtained was not detected and was classified as poor (-5.0 - 5.0 in the filter based kit and 1.5 - 2.0 in the heat treatment) Conclusion: Filter based kit method better than heat treatment method based on LoD and LoQ. Keyword: DNA Isolation, Limit of Detection, Limit of Quantification , Staphylococcus aureus