Transporters are transmembrane proteins that catalyze the passive or active transport of inorganic ions and/or organic solutes across biological membranes. Pre‐steady‐state kinetic methods have been applied to a large number of transporters, including neurotransmitter transporters, sugar transporters, phosphate transporters, and amino acid transporters. The chapter highlights key insights from two case studies: the electrogenic, Na+‐driven secondary‐active transporter for the neurotransmitter glutamate (excitatory amino acid transporter (EAAT)) and the electroneutral Na+‐dependent amino acid exchanger (alanine serine cysteine transporter (ASCT2)). Translocation of the substrate across the membrane is preceded by the binding of the substrate, as well as the ions that potentially drive the transport. A large number of studies have investigated the function of mutated secondary‐active transporters. Analysis of the effects of site‐specific mutations allows the determination of the direct impact of specific amino acid side chains in processes such as ion/substrate binding, as well as conformational changes.