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Summary Synthetic biology often relies on the design of genetic circuits, utilizing “bioparts” (modular DNA pieces) to accomplish desired responses to external stimuli. While such designs are usually intuited, detailed here is a computational approach to synthetic biology design and modeling using optimization-based tools named Eukaryotic Genetic Circuit Design and Modeling. These allow for designing and subsequent screening of genetic circuits to increase the chances of in vivo success and contribute to the development of an application development pipeline. For complete details on the use and execution of this protocol, please refer to Schroeder, Baber, and Saha (2021)., Graphical Abstract, Highlights • Protocol for Eukaryotic Circuit Design (EuGeneCiD) and Modeling (EuGeneCiM) tools • Details modification of these tools for new applications and parallelization • Details reading, formatting, and curation of results for easier understanding • Instructions for combined and separate workflows for EuGeneCiD and EuGeneCiM, Synthetic biology often relies on the design of genetic circuits, utilizing “bioparts” (modular DNA pieces) to accomplish desired responses to external stimuli. While such designs are usually intuited, detailed here is a computational approach to synthetic biology design and modeling using optimization-based tools named Eukaryotic Genetic Circuit Design and Modeling. These allow for designing and subsequent screening of genetic circuits to increase the chances of in vivo success and contribute to the development of an application development pipeline.