Current approaches for rapid assessment of carbon source utilization by whole communities (i.e., community-level physiological profiling or CLPP) provides a limited, biased view of microbial communities with little connection to in situ activities. An alternative CLPP approach based upon fluorometric detection of oxygen consumption may provide a more functionally relevant profile due to reduced selective enrichment resulting from lower substrate concentrations and shorter incubation periods. The approach is based on the fluorescence of an O2-sensitive fluorophore-gel complex loaded on the bottom of microplate wells in response to respiration in the overlying sample (i.e., aqueous soil slurries). Initial optimization studies focused on increasing the sensitivity of the assay by limiting O2 diffusion into the gel-layer via increasing the depth of the liquid layer and by sealing the well from the atmosphere. An agricultural soil and both the organic and mineral layers of a hardwood forest were exposed to four different concentrations of sucrose amendment (100, 50, 10, and 0 ppm). Wells filled with 240 µl vs. 150 µl showed greater response (i.e, higher maximum and integrated fluorescence), reflecting the larger amount of O2 consumption resulting from the higher total amount of carbon added. The 240 µl wells also showed a faster response despite the similar concentrations of added substrate, indicating that O2 diffusion to the gel layer was reduced due to changes in water depth or a deeper layer of settled soil particles on the gel layer. Covering the wells had no effect on the initial rate, but did increase the maximum response and extended the fluorescence. Using the 240 µl volume, sucrose amendments as low as 10 ppm and respiration of endogenous soil organic matter were clearly discernable. Further validation studies are underway in order to define the underlying capabilities/biases of the approach as well as a recommended, standard protocol.