Currently, dominant diagnostic methods in veterinary medicine rely on blood and urine analysis, which come with specific limitations regarding the toxicokinetics of elements and the invasiveness of sample collection. Alternative diagnostic materials (hair, nails) could overcome these limitations. Nowadays, they are not widely used in routine toxicological analysis, mainly due to issues with exogenous contamination and limited sample mass. Furthermore, optimized procedures for determining xenobiotics in these materials are lacking. The main objective of the project was to develop procedures for the determination of Cd and Pb in alternative diagnostic materials (combed hair and mane) collected from horses, and to evaluate their effectiveness in comparison to traditional diagnostic materials such as serum. Hair samples were decontaminated and homogenized with a cryomill before their mineralization. The Cd and Pb were determined in samples after their mineralization by electrothermal atomic absorption spectrometry. The recovery was estimated using certified reference materials. The application of hair as an alternative diagnostic material improved the precision and accuracy of toxicological analysis of Cd and Pb. Further studies should be performed regarding other alternative materials, animal species and heavy metals.