Glucose-6-phosphate dehydrogenase (G6PD) is the rate-limiting enzyme in the pentose phosphate pathway. G6PD is involved in multiple important biochemistry functions, one of which is crucial to generate sufficient NADPH to protect erythrocytes from oxidative stress.  Over 400 million people have G6PD deficiency, mainly in countries where malaria is common ⁽¹⁾. Current assay for G6PD is available but suffers from problems when used with crude preparation in field test.  The mutant forms are normally unstable and may give rise to false positive.  Here, we focus on engineering an active and stable form of E. coli G6PD as a control to be used in the field. A simple high-throughput screening for thermostability was developed using a colorimetric 2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H monosodium salt (WST-8) enzymatic assay. This assay uses the G6PD coupled to a cyclic NADP⁺-NADPH reaction and the tetrazolium, WST-8. The advantage of this method is that, G6PD activity can be measured quantitatively by reading absorbance, as well as qualitatively by estimation with the naked eye. In this study, a variant with enhanced stability, 2-15, and another with enhanced stability and activity, 2-12, were identified. The kinetic properties were studied using a bi-substrate mechanism. The variant 2-12, displayed a 1.5-fold increase in activity than the native. The melting temperature (Tm₅₀) and the half-life was also studied. The native shows a maximum thermostabilty of 60°C for 15 minutes. In contrast, the variants are more stable with an increase of Tm₅₀ by 3-3.5 °C. An enzyme trade-off behavior is shown by the thermostable variant 2-15, where the activity is decreased by 0.5-fold, while still retained approximately 20% of native activity at 60°C for 30 minutes. The assays developed, proved to yield beneficial variants and will allow an economically high-throughput screening for other glucose-producing enzymes.