This study tests the feasibility of using a system of integrated unmanned underwater vehicles (UUVs) and unmanned surface vehicles (USVs) in support of long-endurance oceanographic characterization and acoustic source detection and tracking. Such a system would have the potential to enable long-residence acoustic and oceanographic sampling while providing the opportunity for broad area acoustic coverage and source track resolution with a small number of UUVs using a technique of sequential localization developed by Chan and Towers in 1992. The performance of an underwater glider-based detection system was tested in Monterey Bay using a programmable mobile acoustic source. Multiple subsurface acoustic hydrophones configured similarly to a proposed array of gliders were used to test tracking of the medium frequency source and other performance characteristics. Analysis of recorded Doppler shifts demonstrated closest point of approach (CPA) values consistent with source tracks in both course and speed. A notional sensor layout for the proposed integrated system was demonstrated to show optimal track reconstruction. Factors degrading the ability of this method to make accurate CPA estimates were also explored. The results of this study demonstrate the feasibility of using a system of UUVs/USVs for persistent acoustic sensing and tracking leveraging the characteristics of long-residence time and broad area coverage.