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Thermal inactivation kinetic studies are necessary to determine heat resistances of spores in the development of new thermal processes for low-acid shelf-stable products. Most currently available sample holders used in the kinetic studies take long time to reach the target sample temperature, hence fail to provide isothermal condition. In this research, novel aluminum test cells were developed to facilitate easy loading and unloading solid and liquid food samples in a hermetically sealed 1 ml cavity to evaluate the heat resistance of bacterial spores when heated at temperatures above 100 °C. Design of the test cell was governed by minimum come-up time. A finite element model based on the commercial software ‘FEMLAB’ was used to simulate transient heat transfer and finalize the test cell dimensions. Performance of the new test cell was evaluated against capillary and aluminum thermal death time tube methods in characterizing the heat resistance of Clostridium sporogenes PA 3679 spores in a phosphate buffer and mashed potato at 121 °C. D121 values of PA 3679 spores in both the phosphate buffer and mashed potato using the new test cells were not significantly different (P > 0.05) from those by the capillary tube method. The results indicated that the new test cell is appropriate for studying the inactivation kinetics of bacterial spores in microbial validation of conventional and novel thermal processes for low acid shelf-stable foods.