Abstract Motor proteins are important for transport and force generation in a variety of cellular processes and morphogenesis. Here we design a general strategy for conditional motor mutants by inserting a protease cleavage site at the “neck” between the head domain and the stalk of the motor protein, making the protein susceptible to proteolytic cleavage at the neck by the corresponding protease. To demonstrate the feasibility of this approach, we inserted the cleavage site of TEV protease into the neck of the tetrameric motor Kinesin-5. Application of TEV protease led to a specific depletion and functional loss of Kinesin-5 in Drosophila embryos. By this, we revealed that Kinesin-5 stabilized the microtubule network during interphase in syncytial embryos. The “molecular guillotine” can potentially be applied to many motor proteins due to the conserved structures of kinesin, dynein and myosin with accessible necks. Author summary We design a general strategy for conditional motor mutants by inserting a protease cleavage site between head and stalk domain of the motor protein, making it susceptible to specific proteolytic cleavage. We demonstrate the feasibility of the approach with the motor Kinesin-5 and the protease TEV in Drosophila embryos. This approach can potentially be applied to motor proteins kinesin, dynein and myosin due to the conserved structures.