Focal adhesions (FAs) are specialized structures that link the extracellular matrix (ECM) to actin cytoskeleton, playing essential roles in regulating cell migration and adhesion. Talin is a key protein resided in FAs and serves as a link between integrins and actomyosin II cytoskeleton. Recently, talin has been identified as an A-kinase anchoring protein (AKAP) that binds to protein kinase A (PKA). However, the effects of talin-PKA interaction on FA morphology and dynamics are not fully understood. In this study, a computational tool using FIJI was established to analyze FAs in rat embryo fibroblast cells (REF52s). The effects of disrupted talin-PKA interaction on the number, area, circularity, and aspect ratio of FA were investigated by comparing the point mutation AV1806DD in mouse talin 1 (mTLN1) to wildtype (WT), E1770A mutant, and the double mutant E1770A/AV1806DD. The FA analysis tool enabled accurate quantification of adhesion morphologic properties with raw images and provided insights into the role of PKA in regulating FA formation and turnover through talin interaction. The results of the study demonstrate that disrupted talin-PKA interaction alters FA morphology in REF52s, specifically resulting in a decrease in the total number of FA per cell and a more elongated shape compared to the WT (p