Background: Oxygen minimum zones (OMZS) are expanding in the World Ocean as a result of climate change and direct anthropogenic influence. OMZ expansion greatly affects biogeochemical processes and marine life, especially by constraining the vertical habitat of most marine organisms. Currently, monitoring the variability of the upper limit of the OMZS relies on time intensive sampling protocols, causing poor spatial resolution. Methodology/Principal Findings: Using routine underwater acoustic observations of the vertical distribution of marine organisms, we propose a new method that allows determination of the upper limit of the OMZ with a high precision. Applied in the eastern South-Pacific, this original sampling technique provides high-resolution information on the depth of the upper OMZ allowing documentation of mesoscale and sub mesoscale features (e.g., eddies and filaments) that structure the upper ocean and the marine ecosystems. We also use this information to estimate the habitable volume for the world's most exploited fish, the Peruvian anchovy (Engraulis ringens). Conclusions/Significance: This opportunistic method could be implemented on any vessel geared with multi-frequency echosounders to perform comprehensive high-resolution monitoring of the upper limit of the OMZ. Our approach is a novel way of studying the impact of physical processes on marine life and extracting valid information about the pelagic habitat and its spatial structure, a crucial aspect of Ecosystem-based Fisheries Management in the current context of climate change. © 2010 Bertrand et al.