This work was aimed to evidence that substituted pyridine, 2,6-di-tert-butylpyridine, is a suitable probe for the quantitative investigation of the external acidity in hierarchically structured zeolites. The 2,6-di-tert-butylpyridine was too large to enter the micropores, even in wide pore zeolites, and nearly no sites in nonmesoporous zeolites were available. Accessibility studies of acid sites in zeolites TNU-9 and BEA involving quantitative IR measurements with hindered 2,6-di-tert-butylpyridine as a probe were performed. The extinction coefficients of the 1615 cm-1 diagnostic bands of 2,6-di-tert-butylpyridine interacting with Brønsted acid sites were determined. Lewis acid sites were not detected with the probe. The accessibility factor (AF) for the 2,6-di-tert-butylpyridine probe molecule was defined as the number of sites detected by adsorption of the dTBPy (external sites) divided by the total amount of acid sites in the studied zeolites as quantified by pyridine sorption. Upon desilication resulting in the fabrication of the secondary mesopores, the enhanced accessibility of the protonic sites was observed. In comparison to the mesoporous zeolites with the secondary system of mesopores generated by alkaline leaching, considerably higher accessibility of protonic sites was evidenced in both ultrathin ZSM-5 and delaminated ITQ-2 zeolite.