This work demonstrates a novel compact spectrophotometer, "Mini-spectrophotometer", designed for plasma glucose detection. Unlike conventional spectrophotometer, a light source of the mini spectrophotometer is replaced by a light-emitting diode (LED), and a fabricated polymer-based microwell is used as a cuvette. To validate the downsizing spectrophotometer prototype, the efficiency and reliability for glucose determination are investigated. Using a certain light intensified from LED, the within-run precision of mini-spectrophotometer is found to be 3.9-8.4% while the between-run precision is 6.7-10.8%. The linearity for the quantification of glucose was up to 500 mg dL(-1) and the recovery 99.1 +/- 3.4% is obtained. The sensitive and selective detection of glucose has been observed; with limit of detection (LOD) of 13.5 mg dL(-1) and limit of quantification (LOQ) of 46.2 mg dL(-1), respectively. Hemoglobin and triglyceride at high concentration slightly interferes with the proposed instrument. From comparative studies, there are no significant differences between the glucose concentration measured by mini-spectrophotometer and Shimadzu (r(2) = 0.9862) or CECIL spectrophotometer (r(2) = 0.9853). Using Passing-Bablok regression analysis, the results obtained from mini-spectrophotometer are in close agreement with the two conventional spectrophotometers. Furthermore, using microwell, the sample volume and reagent used in the process can be reduced. The in-house developed mini-spectrophotometer is capable of detecting plasma glucose while maintaining a compact system, demonstrating the potential of high performance, cost-effective, and portable spectrophotometer for clinical chemistry analysis in small routine, research, and teaching medical laboratory technologist. (C) 2018 Elsevier B.V. All rights reserved.