In this study, a modeling software package named UP Finder was developed based on the COBRA toolbox in MATLAB. It facilitated the rapid identification of gene overexpression strategies for the metabolic engineering of targeted overproduction. Gene overexpression targets can be rationally determined by a quantitative evaluation procedure. Development of this interfacial software package was designed to provide the “one-click” convenience, and to facilitate the access for potential users without specific biochemistry and programming backgrounds. By taking advantage of standardized format of SBML models, UP Finder provided broad access for analyzing various targeted products in different microorganisms. Unlike OptForce and FSEOF, UP Finder specifically identified gene targets that were highly related to overproduction rather than all potential important targets. The UPA method used in the UP Finder investigated product yields for each single potential up-regulated pathway by constraining their fluxes with fluxopt, which evaluated their contribution toward theoretical maximum yield and enabled to pinpoint the results as yield/overproduction related. Although some key upstream pathways, such as glycolysis play an important role in improving target product yields when combining with downstream pathway enhancements, the sole overexpression of these genes might not directly contribute to the overproduction.