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Craters of the Moon National Monument and Preserve (CotM) is a strong terrestrial analog for lava tube formations on Mars. The commonality of its basalt composition to Martian lava tubes makes it especially useful for probing how interactions between water, rock, and life have developed over time, and what traces of these microbial communities may be detectable by current flight‐capable instrumentation. Our investigations found that secondary mineral deposits within these caves contain a range of underlying compositions that support diverse and active microbial communities. Examining the taxonomy, activity, and metabolic potential of these communities revealed largely heterotrophic life strategies supported by contributions from chemolithoautotrophs that facilitate key elemental cycles. Finally, traces of these microbial communities were detectable by flight‐capable pyrolysis and wet chemistry gas chromatography‐mass spectrometry methods comparable to those employed by the Sample Analysis at Mars (SAM) instrument aboard the Curiosity rover and the Mars Organic Molecule Analyzer (MOMA) on the upcoming Rosalind Franklin rover. Using a suite of methods for chemical derivatization of organic compounds is beneficial for resolving the greatest variety of biosignatures. Tetramethylammonium hydroxide (TMAH), for example, allowed for optimal resolution of long chain fatty acids. Taken together, these results have implications for the direction of mass spectrometry as a tool for biosignature detection on Mars, as well as informing the selection of sampling sites that could potentially host biosignatures.