Microscopic analysis shows that equine hoof wall is composed of distinct phases of tubular and intertubular horn. The tubule density (TD) of hoof horn is found by ascertaining the number of tubules per unit area and is believed to relate to mechanical properties, resistance to wear, “quality”, hardness and moisture content. The straight grid previously used to ascertain the TD of hoof horn may omit certain tubules on both the inner and outer hoof wall as the wall itself is curved. This present study examined whether there was a significant difference in results for TD between using the conventional straight grid method and a novel method using a curved grid. TD was also compared across three sample sites. An analysis of TD from four equal sections (zones) across the hoof wall was also carried out