Provided herein is a large immuno-sorbent surface area assay (ALISSA) for rapid and sensitive detection of toxin or enzyme activity. This assay is designed to capture a low number of toxin or enzyme molecules and to measure their intrinsic protease activity via conversion of a fluorogenic or luminescent substrate. The ALISSA is significantly faster and more sensitive than methods routinely utilized in the art. This assay is applicable for use for detection of a variety of toxins or enzymes having proteolytic activity, such as botulinum neurotoxin, bacillus anthracis lethal factor, human chitinases, and aspergillus fumigatus proteases. Also provided are methods for constructing and identifying novel luminescent or fluorescent substrates suitable for use with the ALISSA method.