The quantitative evaluation that combining Ratio and Yield parameters improved the relevance of identified pathways for directly leading to the overproduction. In the UP Finder, gene overexpression targets were first identified as up-regulated pathways (the reaction with Ratio > 0) through a flux comparison between the theoretical maximum yield model and the wild-type model. By using fluxopt as a constraint, the contribution of each single identified up-regulated pathway on targeted overproduction was quantitatively evaluated based on the yield simulation. The restriction of Yield > 0 was used to exclude the reactions that are indirectly related to the overproduction, such as the pathways in central carbon metabolism. Thus, the UP Finder provided a quantitative procedure for identifying the key pathways toward overproduction. In addition, it was found in the examples that pathways with higher Ratio values showed closer metabolic distance to the targeted product. Since the greater Ratio value (up-regulation level) indicates the higher demands of metabolic flux for up-regulation to approach the theoretical maximum yield, overexpression of the particular gene would contribute more to the overproduction of targeted products. Therefore, results from the UP Finder not only presented all the key pathway/gene targets related to the overproduction, but the ranking of the outputs with their associated Ratio values also reflected the preference for considering overexpression strategies in pathway design.