A calibrated biosensor was developed to measure mechanical tension across specific proteins in cells with pico-Newton (pN) sensitivity, using single molecule fluorescence force spectroscopy. This sensor was applied to vinculin, a protein that connects integrins to actin filaments and is force-dependent in its recruitment to focal adhesions (FAs). The study revealed that tension across vinculin in stable FAs is approximately 2.5 pN, and that vinculin recruitment and force transmission are regulated separately. Highest 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. Vinculin is required for stabilizing adhesions under force, and these processes can be controlled independently. Focal adhesions (FAs) are complex intracellular linkages between integrins and the F-actin cytoskeleton that transmit and respond to mechanical forces. FAs show complex mechanosensitivity, forming or enlarging when force increases, and shrinking or disassembling when force decreases. However, mechanical forces can also induce FA disassembly, including sliding, a form of controlled disassembly. The development of a genetically encoded vinculin tension sensor with single pN sensitivity allowed for the direct measurement of mechanical tension across vinculin in living cells. The tension sensor module (TSMod) was designed with a 40 amino acid (aa)-long elastic domain derived from spider silk protein flagelliform, which is suitable for measuring pN forces. The TSMod was inserted between the head and tail domains of vinculin after aa 883. Controls included a C-terminally tagged vinculin-venus (VinV) and a tail-less mutant (VinTL), which cannot bind F-actin or paxillin. The sensor was tested in vinculin-deficient cells, where it was properly recruited to FAs, and showed similar FA shape and size to cells expressing VinV. The sensor was also tested for potential confounding factors, such as inter-molecular FRET and effects of vinculin conformation on FRET, and showed that vinculin’s conformational changes do not affect FRET measurements of VinTS. The sensor was used to evaluate responses to cellular forces, showing that VinTS but not VinTL displayed reduced FRET index in FAs on fibronectin, indicating increased mechanical tension. These results were confirmed by fluorescence lifetime microscopy (FLIM), which showed that VinTS displayed significantly longer lifetimes, corresponding to lower FRET efficiency. The sensor was also used to calibrate the tension sensor using single molecule fluorescence-force spectroscopy, revealing that the average force in stationary FAs is approximately 2.5 pN. The study demonstrated that vinculin activation and recruitment to FAs are separable from transmission of force across vinculin. Vinculin is required for FA stabilization under tension, and its failure to bear force isA calibrated biosensor was developed to measure mechanical tension across specific proteins in cells with pico-Newton (pN) sensitivity, using single molecule fluorescence force spectroscopy. This sensor was applied to vinculin, a protein that connects integrins to actin filaments and is force-dependent in its recruitment to focal adhesions (FAs). The study revealed that tension across vinculin in stable FAs is approximately 2.5 pN, and that vinculin recruitment and force transmission are regulated separately. Highest 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. Vinculin is required for stabilizing adhesions under force, and these processes can be controlled independently. Focal adhesions (FAs) are complex intracellular linkages between integrins and the F-actin cytoskeleton that transmit and respond to mechanical forces. FAs show complex mechanosensitivity, forming or enlarging when force increases, and shrinking or disassembling when force decreases. However, mechanical forces can also induce FA disassembly, including sliding, a form of controlled disassembly. The development of a genetically encoded vinculin tension sensor with single pN sensitivity allowed for the direct measurement of mechanical tension across vinculin in living cells. The tension sensor module (TSMod) was designed with a 40 amino acid (aa)-long elastic domain derived from spider silk protein flagelliform, which is suitable for measuring pN forces. The TSMod was inserted between the head and tail domains of vinculin after aa 883. Controls included a C-terminally tagged vinculin-venus (VinV) and a tail-less mutant (VinTL), which cannot bind F-actin or paxillin. The sensor was tested in vinculin-deficient cells, where it was properly recruited to FAs, and showed similar FA shape and size to cells expressing VinV. The sensor was also tested for potential confounding factors, such as inter-molecular FRET and effects of vinculin conformation on FRET, and showed that vinculin’s conformational changes do not affect FRET measurements of VinTS. The sensor was used to evaluate responses to cellular forces, showing that VinTS but not VinTL displayed reduced FRET index in FAs on fibronectin, indicating increased mechanical tension. These results were confirmed by fluorescence lifetime microscopy (FLIM), which showed that VinTS displayed significantly longer lifetimes, corresponding to lower FRET efficiency. The sensor was also used to calibrate the tension sensor using single molecule fluorescence-force spectroscopy, revealing that the average force in stationary FAs is approximately 2.5 pN. The study demonstrated that vinculin activation and recruitment to FAs are separable from transmission of force across vinculin. Vinculin is required for FA stabilization under tension, and its failure to bear force is