As the subtle intricacies of mammalian metabolism have become further elucidated over the past several decades, substantial progress has been made in the medical and scientific communities to address metabolic disorders and the relationships between abnormalities in metabolism at the molecular level and their observable physiological ramifications. The amino acid alanine is known as a key molecule in protein and carbohydrate metabolism and nitrogen regulation, and has further been associated with a number of diseases and pathological conditions. It is also of interest to the commercial and biotechnological industries. Therefore, a rapid and inexpensive procedure for the measurement of alanine in biological or commercial samples would be of great use. Two amperometric methods for the measurement of L-alanine involving enzymatic biosensors are explored. The electrochemical foundations for the operation of the sensors are described. The unique reaction mechanism for the conversion of alanine to measurable products is discussed. Sensors are tested for alanine measurement via the oxidation of hydrogen peroxide and the reduction of oxygen. The response of the sensors to reacted alanine samples as well as standard solutions are evaluated with regards to sensitivity and rapidity. These studies show that the proposed reaction mechanism for alanine conversion and detection is feasible and that the development of an amperometric biosensor for this purpose is an attainable goal, while also suggesting that optimizing the sensitivity of the sensors is paramount to its utility