An objective dietary biomarker would help clarify the contribution of sugar-sweetened beverage (SSB) intake to obesity and chronic disease risk. Previous studies have proposed the carbon isotope ratio (δ(13)C) as a biomarker of SSB intake but found associations that were of modest size and confounded by other components of the diet. We investigated whether the δ(13)C values of nonessential amino acids (δ(13)CNEAA) in RBCs could provide valid biomarkers that are more specific to SSBs. We assessed the associations of RBC δ(13)CNEAA with SSB intake in a study population of 68 Yup'ik people, using gas chromatography/combustion/isotope ratio mass spectrometry to measure δ(13)CNEAA and four 24-h dietary recalls to assess intake. Among RBC nonessential amino acids, alanine δ(13)C (δ(13)Calanine) was strongly correlated with intake of SSBs, added sugar, and total sugar (r = 0.70, 0.59, and 0.57, respectively; P < 0.0001) but uncorrelated with other dietary sources of elevated δ(13)C. We also evaluated whether sweetener intake could be noninvasively assessed using hair δ(13)Calanine in a subset of the study population (n = 30). Hair δ(13)Calanine was correlated with RBC δ(13)Calanine (r = 0.65; P < 0.0001) and showed similar associations with SSB intake. These results show that δ(13)Calanine in RBCs provides a valid and specific biomarker of SSB intake for the Yup'ik population and suggest RBCs and hair δ(13)Calanine as candidate biomarkers of SSB intake for validation in the general U.S. population. Ultimately, these biomarkers could clarify our understanding of whether and how SSB intake contributes to chronic disease.