Background Substrate accessibility remains a key limitation to the efficient enzymatic deconstruction of lignocellulosic biomass. Limited substrate accessibility is often addressed by increasing enzyme loading, which increases process and product costs. Alternatively, considerable efforts are underway world-wide to identify amorphogenesis-inducing proteins and protein domains that increase the accessibility of carbohydrate-active enzymes to targeted lignocellulose components. Results We established a three-dimensional assay, PACER (plant cell wall model for the analysis of non-catalytic and enzymatic responses), that enables analysis of enzyme migration through defined lignocellulose composites. A cellulose/azo-xylan composite was made to demonstrate the PACER concept and then used to test the migration and activity of multiple xylanolytic enzymes. In addition to non-catalytic domains of xylanases, the potential of loosenin-like proteins to boost xylanase migration through cellulose/azo-xylan composites was observed. Conclusions The PACER assay is inexpensive and parallelizable, suitable for screening proteins for ability to increase enzyme accessibility to lignocellulose substrates. Using the PACER assay, we visualized the impact of xylan-binding modules and loosenin-like proteins on xylanase mobility and access to targeted substrates. Given the flexibility to use different composite materials, the PACER assay presents a versatile platform to study impacts of lignocellulose components on enzyme access to targeted substrates.