Pyrite is an important mineralogical component of most sulphide ore deposit classes, where it commonly forms part of the gangue mineralogy, but may also represent an important ore mineral (i.e., auriferous pyrite). Effective and efficient separation of pyrite is thus a crucial step during most ore processing operations, and this is in part influenced by the pyrite mineral chemistry. Here, electrochemical measurements were used to study the reactivity of a series of well-characterised synthetic trace-element substituted pyrite samples under alkaline conditions relevant to industrial flotation. The presence of metals and metalloid impurities (As, Au, Co, and Ni) in pyrite were tested using rest potential measurements to infer oxidation and associated hydrophobicity. In the absence of any collector phases, pure- and Ni-substituted pyrite have the highest rest potential, followed by Co-substituted pyrite and couple-substituted (Co + Au) pyrite, whilst As-substituted pyrite has the lowest measured rest potential. Importantly, the degree of oxidation at the mineral surface correlates linearly with the concentration of each of the substituents, with the largest effect observed when As is the substituent. These results correspond to the semiconducting properties and noble character of each pyrite sample, with n-type pyrite (Au-, Co- and Ni-substituted) associated with noble character and high rest potential, whereas p-type As-substituted pyrite associated with least noble character and lowest rest potential. With the addition of a potassium amyl xanthate collector, the mineral chemistry further had an impact on the probability of dixanthogen formation. Increased substituent concentration in the pyrite lattice decreased the probability of dixanthogen formation, except in a sample where high Au (and moderate Co) was incorporated. These results highlight the importance of developing improved understanding of the impacts of substitution mechanisms on the surface reactivity and flotability of pyrite. Such an understanding will form the foundation for further improved (and engineered) approaches towards reagent design and mixture. This will serve to optimise separation of both gangue and valuable pyrite by using fundamental knowledge to target specific collector bands and flotation domains.