Studying metabolism may assist in understanding the relationship between normal and dysfunctional mitochondrial activity and various diseases, such as neurodegenerative, cardiovascular, autoimmune, psychiatric, and cancer. Nuclear magnetic resonance-based metabolomics represents a powerful method to characterize the chemical content of complex samples and has been successfully applied to studying a range of conditions. However, an optimized methodology is lacking for analyzing isolated organelles, such as mitochondria. In this study, we report the development of a protocol to metabolically profile mitochondria from healthy, tumoral, and metastatic tissues. Encouragingly, this approach provided quantitative information about up to 45 metabolites in one comprehensive and robust analysis. Our results revealed significant differences between whole-cell and mitochondrial metabolites, which supports a more refined approach to metabolic analysis. We applied our optimized methodology to investigate aggressive and metastatic breast cancer in mouse tissues, discovering that lung mitochondria exhibit an altered metabolic fingerprint. Specific amino acids, organic acids, and lipids showed significant increases in levels when compared with mitochondria from healthy tissues. Our optimized methodology could promote a better understanding of the molecular mechanisms underlying breast cancer aggressiveness and mitochondrial-related diseases and support the optimization of new advanced therapies.