Much is still unknown about why freshwater mussels (Unionidae) are particularly sensitive to environmental change. A better understanding of freshwater mussel metabolism is needed, and the field of environmental metabolomics holds the promise to inform these questions. A number of protocols exist for the extraction of metabolites for identification from animal tissues. As a first step in the application of environmental metabolomics to the study of freshwater mussels, we compared extraction protocols using an inorganic oxidizing acid (perchloric acid), an organic nitrile (acetonitrile), and a salt/water solution (Ringer's solution) to establish an uncomplicated, robust, repeatable and inexpensive tissue extraction protocol for freshwater mussel tissue. Perchloric acid resulted in notable extraction of energy-related nucleotides (AMP/ADP/ATP), yet had the lowest peak count of the three extraction methods and showed poor repeatability. Acetonitrile and Ringer's solution yielded metabolite extraction results similar to each other with Ringer's solution having the greatest number of peaks particularly in the 3.0–4.5 ppm sugar/amino acid range. Ringer's solution is simple to use, safe and consistent and bears consideration when selecting an extraction protocol for ¹H nuclear magnetic resonance experiments.