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In the treatment of human cancer, the existing drug response identification and assessment methods have some issues, such as high cost, slow processing, and low accuracy. Responsive methods that easily and indirectly assess patient tissue response to drugs or small molecules offer promising options for improving drug safety, and they are able to identify the right treatment for specific patients. Digital image correlation is a good technique that detects a part of the image between two images. In this article, a new method of correlation called significant improvement of correlation (SIC) was proposed. Data were taken from the confocal microscopy of two‐photon excited fluorescence (TPEF) probe images taken every 5 min for short‐term monitoring (STM) and long‐term monitoring (LTM; 12, 24, and 48 hours). Cells were cultured, and a new compound TPEF was tested and injected the drug. The effect of drug on …