Intraflagellar transport (IFT) is essential for the assembly, maintenance and function of most eukaryotic cilia. It acts as a bidirectional cargo transport system, powered by kinesin and dynein motors to navigate through the crowded ciliary environment. Here, we describe a method to isolate intact IFT trains from live cells and reconstitute their motility on synthetic microtubules in vitro. This system enables the controlled investigation of the biophysical properties of IFT trains, including their sensitivity to tubulin post-translational modifications. With minor adaptations, this approach can be extended to study other intracellular transport processes.