This study develops a calibrated biosensor to measure mechanical forces across specific proteins in cells with pico-Newton (pN) sensitivity. The sensor, a genetically encoded tension sensor (VinTS) for vinculin, a protein that connects integrins to actin filaments, is designed to measure forces in focal adhesions (FAs). The sensor's sensitivity is demonstrated through single molecule fluorescence force spectroscopy, showing that tension across vinculin in stable FAs is approximately 2.5 pN. The study reveals that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. High tension is associated with adhesion assembly and enlargement, while low tension is observed in disassembling or sliding FAs at the trailing edge of migrating cells. Additionally, vinculin is required for stabilizing adhesions under force, highlighting that both processes can be controlled independently. These findings provide new insights into the regulation of FA dynamics and mechanotransduction.This study develops a calibrated biosensor to measure mechanical forces across specific proteins in cells with pico-Newton (pN) sensitivity. The sensor, a genetically encoded tension sensor (VinTS) for vinculin, a protein that connects integrins to actin filaments, is designed to measure forces in focal adhesions (FAs). The sensor's sensitivity is demonstrated through single molecule fluorescence force spectroscopy, showing that tension across vinculin in stable FAs is approximately 2.5 pN. The study reveals that vinculin recruitment to FAs and force transmission across vinculin are regulated separately. High tension is associated with adhesion assembly and enlargement, while low tension is observed in disassembling or sliding FAs at the trailing edge of migrating cells. Additionally, vinculin is required for stabilizing adhesions under force, highlighting that both processes can be controlled independently. These findings provide new insights into the regulation of FA dynamics and mechanotransduction.