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HIF-1α is regarded as a target for drug development in several diseases such as cancer. For high throughput screening of HIF-1α-targeted drug, we need to examine the activity quantitatively. In the present study, we proposed a method where stable expression system of HIF-1α was combined with image correlation analysis. When the stable transformants were labeled with DRAQ5, we could detect Co2+-induced nuclear translocation by the use of cross-correlation analysis of the dual labeling images. In the case of high throughput screening for HIF-1α-targeted drug, we should use Pearson's correlation coefficient to judge nuclear translocation.