Anaerobic co-digestion (AcoD) can increase methane production of anaerobic digesters in plants treating wastewater sludge by improving the nutrient balance needed for the microorganisms to grow in the digesters, resulting in a faster process stabilization. Substrate mixture proportions are usually optimized in terms of biogas production, while the metabolic biodegradability of the whole mixture is neglected in this optimisation. In this aim, we developed a strategy to assess AcoD using metabolomics data. This strategy was explored in two different systems. Specifically, we investigated the co-digestion of wastewater sludge with different proportions of either grass or fish waste using untargeted High Performance Liquid Chromatography coupled to Mass Spectrometry (HPLC-MS) metabolomics and chemometrics methods. The analysis of these data revealed that adding grass waste did not improve the metabolic biodegradability of wastewater sludge. Conversely, a synergistic effect in the metabolic biodegradability was observed when fish waste was used, this effect being the highest for 25% of fish waste. In conclusion, metabolomics can be regarded as a promising tool both for characterizing the biochemical processes occurring during anaerobic digestion, and for providing a better understanding of the anaerobic digestion processes.