Transketolases (TKs) are ubiquitous thiamine pyrophosphate (TPP)-dependent enzymes of the nonoxidative branch of the pentose phosphate pathway. They are considered as interesting therapeutic targets in numerous diseases and infections (e.g., cancer, tuberculosis, malaria), for which it is important to find specific and efficient inhibitors. Current TK assays require important amounts of enzyme, are time-consuming, and are not specific. Here, we report a new high throughput electrochemical assay based on the oxidative trapping of the TK-TPP intermediate. After electrode characterization, the enzyme loading, electrochemical protocol, and substrate concentration were optimized. Finally, 96 electrochemical assays could be performed in parallel in only 7 min, which allows a rapid screening of TK inhibitors. Then, 1360 molecules of an in-house chemical library were screened and one early lead compound was identified to inhibit TK from E. coli with an IC50 of 63 μM and an inhibition constant (KI) of 3.4 μM. The electrochemical assay was also used to propose an inhibition mechanism.