The membrane bound enzyme monoamine oxidase exist in two splice variants designated A and B (MAO-A and MAO-B) and are key players in the oxidative metabolism of monoamines in mammalians. Despite their importance and being a prevalent target for the development of inhibitors as drugs, no systematic study of substrate specificity has been reported. In this study we present a systematic study of the MAO-A and MAO-B substrate specificity profile by probing two series of phenethylamine analogs. Km and kcat values were determined for four N-alkyl analogs 2–5 and four aryl halide analogs 6–9 at MAO-A and MAO-B. A following in silico study disclosed a new adjacent compartment to the MAO-B substrate pocket defined by amino acids Tyr188, Tyr435, Tyr398, Thr399, Cys172 and Gly434. This new insight is important for the understanding of the substrate specificity of the MAO-B enzyme and will be relevant for future drug design within the field of monoamines.