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Summary: Assaying enzymatic degradation of water-insoluble substrates like cellulose is challenging because only the substrate surface is accessible to the enzymes resulting in low reaction rates. Here, we describe a protocol for surface pitting observation technology (SPOT), an ultra-sensitive quantitative assay for analyzing enzymatic hydrolysis of cellulose. We describe the use of a porous substrate to accelerate the hydrolysis rate of cellulose. We also detail the steps for combining inkjet patterning and optical profilometry to analyze volume loss upon hydrolysis.For complete details on the use and execution of this protocol, please refer to Tsudome et al. (2022).1 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.