Diabetes in general can be classified as Type 1, Type 2, gestational diabetes mellitus that occur during pregnancy, and specific types of diabetes due to certain health condition. Monitoring glucose level is crucial in order to maintain a healthy lifestyle, especially among diabetic patients. Today, researchers looking for a less painful, cheap, and user-friendly noninvasive method to monitor the body glucose level. One of the promising approaches is sweat based glucose detection. In order to calibrate the sensing device, we determined the limit of detection of the glucose concentration in sweat liquid by the well-established enzymatic reaction. This paper will discuss the utilisation of a microplate reader to measure the sensitivity of the chromogenic change in reaction. 50μL of glucose oxidase-peroxidase (GOD-POD) enzyme and 1.67μL glucose sample with different concentrations were mixed in 96 well flat bottom microplate, and the absorbance were detected by using a microplate reader. The absorbance value was used to calculate the concentration. The calculated concentration was compared with the known concentration. From the experimental procedure, the results showed the consistency of calculated and known concentration and R2 = 0.9995