Abstract: Micro-scale thermophoresis (MST) is a newly developing biophysical technique for the detection of molecular interaction, which has the advantages of low sample consumption, short detecting times and wide range of applications. However, in actual operation, several experimental conditions and factors must be considered, such as the fluorescent labeling of target molecules, the quality control of samples and the selection of sample concentrations. It not only affects the quality of MST data but may also results in significant deviations in measured affinities. On the basis of a brief outline of technical principle of MST, the critical experimental conditions and elements in MST test were sorted out. By reasonable selection and optimization of the experimental conditions of MST, it helps experimentalists to obtain the actual and valid MST data and affinity results.