Research for possible biosignature gases on habitable exoplanet atmospheres is accelerating, with observations possible with next-generation telescopes. We explore isoprene, C5H8, as a biosignature gas, motivated by isoprene’s substantial production rate on Earth (rivaling that of methane at ~ 500 Tg yr-1). Furthermore, because isoprene’s geochemical formation is highly thermodynamically disfavored at temperate terrestrial planet conditions, isoprene has no known abiotic false positives. We model the photochemistry and spectroscopic detectability of isoprene in habitable temperature, rocky exoplanet anoxic atmospheres with a variety of atmosphere compositions under different host star UV fluxes. We focus on anoxic atmospheres because in Earth’s oxygen-rich environment isoprene is rapidly destroyed by oxygen-containing radicals. We find that isoprene can only accumulate to detectable levels if it enters a “runaway” phase whereby the production rate surpasses the destruction rate, controlled by the flux of UV-destructive photons. In such a situation, isoprene would be detectable with 20 JWST transits for the most favorable exoplanet scenario, a super-Earth sized exoplanet transiting an M dwarf star with a H2-dominated atmosphere. In this situation the simulated isoprene column-averaged mixing ratio is on order of 100 ppm, ~ 100 times Earth’s average production rate (which does occur in niche environments on Earth). One caveat is that isoprene’s 3–4 micron spectral feature is hard to distinguish from that of methane’s. Despite the challenges, isoprene is worth adding to the menu of potential biosignature gases: isoprene production is ubiquitous to a diverse array of evolutionary distant organisms, from bacteria to plants and animals—few, if any volatile secondary metabolites have a larger evolutionary reach.