Protein assembly is a crucial process in biology, because most proteins must assemble into complexes to perform their function in the cell. The mitochondrial Tim9-Tim10 translocase complex, located in the mitochondrial intermembrane space, plays an essential chaperone-like role during the import of mitochondrial membrane proteins. The complex consists of three molecules of each subunit arranged alternately in a ring-shaped structure. While structural and functional studies have indicated a dynamic nature of the complex, little is known about the assembly process and the mechanism of its function. Here we investigated the assembly process of yeast Tim9-Tim10 complex in real time, using stopped-flow fluorescence coupled with Trp mutagenesis, and stopped-flow light scattering techniques. We show that different parts of the proteins are assembled at different rates; also assembly intermediates consisting four subunits arise transiently before formation of the final hexameric Tim9-Tim10 complex. Interestingly, the assembly intermediate has more organised N-terminal helices that form an inner layer of the complex, but not the C-terminal helices, which form the outer layer of the complex. In addition, using analytical ultracentrifugation techniques, we show that Tim9 forms a homo-dimer while Tim10 is a monomer. A four-step assembly pathway of Tim9-Tim10 complex, involving formation of hetero-dimer and tetramer assembly intermediates, is proposed. This study provides the first description of the assembly pathway of this translocase complex, and insight into the mechanism of its function.