This protocol provides a comprehensive guide for constructing high-quality genome-scale metabolic reconstructions, which are essential for systems biology studies. The process involves four main stages: creating a draft reconstruction, manual refinement, conversion to a mathematical model, and network evaluation. Each stage includes detailed steps and common challenges, such as data curation, reaction validation, and model debugging. The protocol emphasizes the importance of organism-specific information, experimental data, and literature reviews to ensure the accuracy and reliability of the reconstructed networks. It also highlights the need for iterative refinement and validation to improve the predictive potential of the models. The protocol aims to standardize the reconstruction process, making it more accessible and reproducible, and to enhance the quality and comparability of metabolic reconstructions across different organisms.This protocol provides a comprehensive guide for constructing high-quality genome-scale metabolic reconstructions, which are essential for systems biology studies. The process involves four main stages: creating a draft reconstruction, manual refinement, conversion to a mathematical model, and network evaluation. Each stage includes detailed steps and common challenges, such as data curation, reaction validation, and model debugging. The protocol emphasizes the importance of organism-specific information, experimental data, and literature reviews to ensure the accuracy and reliability of the reconstructed networks. It also highlights the need for iterative refinement and validation to improve the predictive potential of the models. The protocol aims to standardize the reconstruction process, making it more accessible and reproducible, and to enhance the quality and comparability of metabolic reconstructions across different organisms.