Objectives In order to establish DIAAS in humans, the FAO recommended to develop a new method to measure indispensable amino acid (IAA) digestibility. This method uses two isotopic labeling, one for the protein to test and one for a reference protein. Spirulina was chosen as the 13C reference protein due to its commercial availability and affordability. However, the real digestibility of spirulina protein has not been measured in vivo. This work aims to assess the digestibility of spirulina and its repeatability in different meal tests in rats. Methods 23 Wistar male rats were fed a test meal containing 0.5 g of 15 N protein from either spirulina (n = 7), sunflower n = 8) or goat milk isolate (n = 8) and 10 mg of 13C labeled spirulina. Rats were euthanized 6 h after the meal and their digestive luminal contents (stomach, small intestine, ileum, caecum, colon) were collected. Protein digestibility was determined for the test and the reference proteins by measuring 15 N and 13C enrichments in the digesta by EA-IRMS. Caecal IAA digestibility of 13C spirulina was determined by measuring the quantity of AA in the caecum by UPLC and the 13C enrichment in AA by GC-C-IRMS. Group effects were tested using one way ANOVA and differences between groups using Bonferroni test. Results Six hours after ingestion, most of the dietary 15 N and 13C were found in the caecum and colon. But there at least twice more 15 N nitrogen in the caecum and colon in the spirulina group than in the two other groups. Therefore, spirulina protein digestibility (86.0 ± 0.7%) was lower (P < 0.001) than sunflower (95.1 ± 0.5%) and goat milk digestibility (97.2 ± 0.2%). 13C spirulina digestibility tended to be different (P = 0.06) when mixed to spirulina (90.6 ± 0.6%), sunflower (88.8 ± 0.5%) or goat milk (89.0 ± 0.5%) isolates. The caecal IAA digestibility of 13C spirulina was lower in the spirulina group than in sunflower and goat milk groups for every IAA tested, and the mean was 91.6 ± 0.2% for sunflower, 91.4 ± 0.4% for goat milk and 85.4 ± 0.6% for spirulina. Conclusions Spirulina protein is of lower digestibility than other animal or plant proteins. Protein and amino digestibility of a tracer dose of 13C spirulina appears to vary depending on the protein component of the meal. These results question the use of spirulina as a reference protein for the dual isotope method. Funding Sources French Research National Agency (ANR), SOFIPROTEOL. Supporting Tables, Images and/or Graphs