Carnosine is an endogenous di-peptide (b-alanine -L- histidine) involved in maintaining tissue homeostasis. It is most abundant in skeletal muscle where its concentration has been determined in biopsy samples using tandem mass spectrometry (MS-MS). Carnosine levels can also be assessed in intact leg muscles by proton magnetic resonance spectroscopy (1H-MRS) or in blood and urine samples using mass spectrometry. Nevertheless, it remains uncertain how carnosine levels from these distinct compartments are correlated with each other when measured in the same individual. Furthermore, it is unclear which measurement modality might be most suitable for large-scale clinical studies. Hence, in 31 healthy volunteers, we assessed carnosine levels in skeletal muscle, via 1H-MRS, and in erythrocytes and urine by MS-MS. While muscle carnosine levels were higher in males, there was no sex-associated difference in urinary or erythrocyte levels. In a linear regression model adjusted for age, sex, race, and diet, there was a positive association between erythrocyte and urinary carnosine. However, no association was observed between 1H-MRS and erythrocytes or urinary measures. In the relationship between muscle versus urinary and erythrocyte measures, females had a positive association, while males did not show any association. We also found that 1H-MRS measures were highly sensitive to location of measurement. Thus, it is uncertain whether 1H-MRS can accurately and reliably predict endogenous carnosine levels. In contrast, urinary and erythrocyte carnosine measures may be stable and in greater synchrony, and given financial and logistical concerns, may be a feasible alternative for large-scale clinical studies.