Delta smelt Hypomesus transpacificus is a Critically Endangered fish native to the San Francisco Estuary. Respirometry methods quantify O 2 consumption (MO 2 ) to estimate metabolic activity, providing insights on organismal energetic budgets critical for devising conservation strategies. Due to its widespread adoption and utility, respirometry protocols have been established to foster quality control. However, delta smelt respirometry attempts over the past 12 yr have been largely unsuccessful, and high mortality rates during tests were only circumvented with very short measurement duration. This prevented the measurement of routine metabolic rate (RMR) and critical O 2 tension (P crit ) and obstructed delta smelt physiological research and conservation. Here, we detailed the anecdotal, behavioral, and empirical evidence that culminated in successful delta smelt RMR and P crit measurements at 10, 12, 15, 17 and 19°C. We discovered the delta smelt is physostomous and requires frequent air bladder refills to maintain buoyancy and found the inclusion of an air pocket within their respirometry chamber greatly improved measurements. Daytime respirometry measurements yielded greater survival compared to overnight measurements; time of day did not cause significant RMR differences. Moreover, fasting did not significantly influence MO 2 . Delta smelt RMR values at 10 and 12°C were significantly lower than fish acclimated to 15, 17, and 19°C. Delta smelt P crit showed a similar pattern, but only the 19°C treatment showed significantly higher P crit level. Altogether, this methodology allows for acquisition of critical knowledge needed for delta smelt conservation research, reducing conservation aquaculture operating costs, and improving survival during transport and field supplementation.