Background: 96-well microtiter plate assay are becoming popular analytical procedures in laboratory and clinical practices generating a demand for microtiter plate readers. The present colorimetric and fluorimetric based microtiter plate reader are efficient in general applications such as measuring reflectance, absorbance, and optical density of reaction mixtures. However, a dedicated system incorporating automation and computational techniques in specific microtiter plate readers for Drug discovery and Microbial viability, provides portability and compatibility with highthroughput analysis at affordable cost. Objective: To develop image-based reader system for semi-quantitative measurements of transport assay performed in microtiter plate. In this work, we focus on deduction of illumination source and classification of fluorescence emitted by transport assay using this microtiter plate reader. Method: We have taken the images of 96-well fluorescence efflux assay and developed an appropriate image analysis system to read the individual well images and categorize the results as fast efflux and slow efflux by the classifier. The images exhibited non-uniform illumination due to UV transilluminator, which then replaced by the circuit of blue LED after performing a study on its classification performance.