Companion animals may receive all their nutrition from commercially-prepared pet foods and responsible food manufacturers ensure their diets are nutritionally balanced and complete. The current recommendations for dietary methionine, an essential amino acid (EAA) often found in limited amounts in raw materials, is however based upon limited evidence (1). The indicator amino acid oxidation (IAAO) method assesses EAA requirements by monitoring the rate of amino acid oxidation of a 13C-labelled EAA (the indicator). This is based on the principle that if an amino acid (AA) is rate limiting for protein synthesis, then other AAs present in excess are oxidised (2). This rate of oxidation is measured from the appearance of 13CO2 in the breath, derived from, in this case, 1-13C phenylalanine added to the diet. By providing different amounts of methionine in the diet and measuring the rate of appearance of 13CO2 from the indicator AA, breakpoint analysis on a two-phase linear regression can be used to identify the minimum amount of methionine required to overcome limited substrate availability for protein synthesis. The aim of this study was to improve the IAAO method used previously (3) by reducing variability, using fewer dogs, acquiring fewer breath samples and studying fewer diets. After obtaining all required ethical approvals, thirteen adult dogs maintained at a normal body condition score, were fed 5 semi-purified diets: one basal and four test diets each varying in methionine concentration (1.7g and 0, 0.3, 0.6 and 1.5 g/1000kcal, respectively). Each week the dogs were fed the basal diet (1 meal/day) for 4-days, followed by 3-days on a test diet (2-days with 4 meals/day and 1-day with 8 meals/day- the study day). All dogs received each test diet 3 times. On the study day (after 6 months of animal training) meals were given hourly and breath samples were collected prior to tracer dosing and during the last 2 hours. The daily phenylalanine requirement was provided with the first meal. Meal 4 contained a priming dose of 1-13C phenylalanine, with “top-ups” added to each subsequent meal. Breath was collected using a respiratory mask and collection bags. The ratio of 13CO2 :12CO2 in the breath was measured, by continuous flow isotope ratio mass spectrometry to calculate the rate of appearance of 13CO2. Breakpoint analysis estimated a minimum methionine requirement of 0.337g/1000kcal with 95% CI (0.330, 0.345). This value is similar to previous estimates (3), but with greater precision (±2.2% vs. 27.1%). This approach reduced the numbers of dogs needed (12 vs. 21) whilst requiring less sampling points (7 vs. 11) and fewer diets to be tested (4 vs. 9). Altering the diet to an extruded pellet (vs. mash (3)) facilitated the development of a method with more robust estimates of EAA requirements in dogs, which will contribute to improving canine diets.