The indirect estimation of thiamine levels in human blood by measuring thiamine pyrophosphate effect on erythrocyte transketolase activity is the method of choice in most clinical laboratories. We describe here an optimized, time-saving, and accurate method to determine the thiamine pyrophosphate effect in as many as 16 blood samples simultaneously. The method is based on a multi-point determination using a computer remote-controlled microplate reader. For multiple sample handling, three pooled reaction mixtures are freshly prepared and loaded onto a 96 well microtitre plate. A pre-written software is then initiated to remote-control the system. The data is retrieved and processed to calculate thiamine pyrophosphate effect by a self-written "macro" on a "Quattro-Pro" worksheet database. This method proves to be highly accurate (coefficient of variance: 2.7%), reproducible (coefficient of variance: 4.1%) and economical.