In equine medicine the use of Botox® is experimental. Dosages are determined from human treatment-protocols and limited numbers of equine studies. Determination of target-muscle volume can be helpful to extrapolate human dosages. The aim of the study was to calculate a formula enabling the estimation of the deep digital flexor muscle (DDFM) volume based on distances between anatomical landmarks. Nineteen cadaveric limbs were collected and distance A (top of olecranon to Os carpi accessorium) and B (circumference of limb) were measured. Converting mathematical formulas, C was calculated: π × (((0.5B)/π)(2)) × A. DDFM volume was determined by water displacement. Linear Regression Analysis was used to analyse data. The line best fitting the observed points was: Ln(volume[ml]) = -1.89 + 0.98 × Ln(value C[cm(3)]). Correlation was highest when natural logarithm was applied to both variables and was 0.97. The calculated formula enables estimating DDFM volume of a living horse. This estimated volume can be useful to apply human Botox® treatment-protocols.