An accessibility index (ACI) was derived from infrared spectroscopy of substituted alkylpyridines with different size (pyridine: 0.57 nm, 2,6-lutidine: 0.67 nm, and 2,4,6-collidine: 0.74 nm) over hierarchical porous ZSM-5 crystals. The samples were prepared by selective silicon extraction of a commercial sample in NaOH (desilication), and they contained different degrees of intracrystalline mesoporosity. Our results demonstrate the enhanced accessibility of Brønsted acid sites in the hierarchical zeolites. For example, a relatively bulky molecule such as collidine, which probes practically no acid site of the parent medium-pore MFI structure, can access up to 40% of the acid sites in the mesoporous sample. The ACI is a powerful tool to standardize acid site accessibility in zeolites, and can be used to rank the effectiveness of synthetic strategies to prepare hierarchical zeolites.