We demonstrated that the indicator amino acid oxidation (IAAO) method could be employed for the evaluation of quality of dietary protein by comparing the protein intakes required to meet metabolic demand in rats fed different proteins. The objective of this study was to validate a simple evaluation method for determining the quality of dietary protein using the IAAO technique. Male Sprague-Dawley rats (5-6 wk old) were fed meals composed of graded protein, using either casein, wheat gluten (WG), soy protein isolate (SPI), or egg white protein (EW), every 3 h from 09:00 to 18:00. Administration of L-[1-¹³C]phenylalanine was performed hourly from 15:00 to 18:00. The ¹³CO₂ level in breath CO₂ was measured at 18:30. The protein intake values required to meet the metabolic demand based on the breath ¹³CO₂ data for the dietary casein, WG, SPI, and EW intake were 18.0, 22.2, 17.5, and 10.1 g/kg BW/d, respectively. The breath ¹³CO₂ concentrations corresponding to the protein intake of 7.5 g/kg BW/d for casein, WG, SPI, and EW were 9.8, 10.9, 10.3, and 8.9 (‰)/100 g BW, respectively. A significant correlation was demonstrated between the protein intake required to meet the metabolic demands and the ¹³CO₂ concentration in the breath for a protein intake of 7.5 g/kg BW/d (r=0.967; p<0.05). These results demonstrated that the protein intake required to meet metabolic demand could be estimated and that the quality of the dietary protein could be evaluated using the ¹³CO₂ concentration in the breath with a protein intake of 7.5 g/kg BW/d.