The characterization of acid rock drainage (ARD) is traditionally based on mineralogical and geochemical techniques (e.g., Acid Base Accounting tests). The complexity of ARD processes warrants contribution of methods from various disciplines. In the past decade, the increasing role of environmental isotopes in pollution monitoring has enabled the successful application of isotope methods in ARD investigations. While isotopic compositions of different pollutants can refer to their parent mineral, the degree of isotope fractionations are indicative of the mechanisms taking place during the release and transportation of ARD-related contaminants. In natural environments, however, the measured isotope fractionations are predominantly the result of several coexisting or sequential processes. Therefore, the identification and quantification of the distinct contributions of these processes to isotope variations is difficult and requires well-defined laboratory conditions, where the influence of ARD generation on different isotope systems can be assessed with greater certainty. This review provides readers with a single source of information regarding isotopic variations generated by laboratory pyrite leaching.