Kinesins transport numerous cargoes throughout the cell, and the accurate long-distance transport and sorting of cargoes is particularly important in highly polarised cells such as neurons. Different approaches have been developed to address which cargoes are transported by which kinesin, including the split-kinesin method; however, it requires the use of single-cell imaging, restricting its broader use. In this ‘Tools and Resources’ article, Jessica Hummel and Casper Hoogenraad (Hummel and Hoogenraad, 2021) now expand on the split-kinesin method and present an adaptable toolbox of engineered kinesins to manipulate polarised trafficking of cargoes in neurons by chemical induction. To demonstrate the usefulness of their approach, the authors first screen a library of kinesin tail domains for an interaction with a specific cargo, before utilising the system to employ a specific kinesin tail domain to screen for interactions with a variety of cargoes, including organelles and vesicles. Furthermore, they examine in more detail the interactions of KIF16B with dense core vesicles (DCVs) and endosomes, and show that its PX domain is sufficient for interactions with both structures. Moreover, the method can also be used to artificially target a cargo to a different neuronal compartment in cultured neurons, even overriding their inherent polarised cargo sorting. This approach thus provides a powerful tool to identify, quantify and study kinesin–cargo interactions in living neurons.