The giant protein titin contributes to muscle force generation. However, titin's mechanical function is still incompletely understood, in part because no tool has been available to specifically cleave the titin springs in situ. In a new genetic mouse model, a tobacco etch virus (TEV) protease-recognition site and a HaloTag were cloned into elastic titin. The HaloTag-TEV cassette allowed for in-situ imaging of titin, specific proteolysis during myofiber mechanics and visualization of successful cleavage. Using permeabilized skeletal myofibers homozygous for HaloTag-TEV titin (knock-in; KI), we measured passive force over the sarcomere-length (SL) range 2.2-3.4 μm and Ca2+-triggered force (pCa 5.0) at 2.6 μm SL, in the absence or presence of TEV enzyme. Wildtype (WT) myofibres treated with TEV protease served as controls.Incubation of myofibers with TEV protease cleaved HaloTag-TEV titin specifically and completely in <20 min (room temperature), but had no effect on other proteins and on titin in WT myofibers. Titin cleavage affected myofiber ultrastructure (measured by electron microscopy) only little in the absence of a mechanical perturbation. However, mechanical stressing of TEV-treated KI fibers caused sarcomere destabilization. Both the Z-discs and the myosin A-bands became disordered. Passive tension dropped with titin-cleavage, on average by ∼70%. The remaining tension was attributed to other cytoskeletal structures/extracellular-matrix proteins. Active force at pCa 5.0 was reduced by titin-cleavage, on average by ∼50% vs. controls. We conclude that this model allows for the reliable quantitation of the contribution of titin-based force to passive and active muscle tension. We find that titin's spring force is not only necessary for most of the passive tension generated by skeletal muscles, but also for the regular structure of all sarcomere bands and for maintaining high Ca2+-dependent tension.