Background: The iodine status of the US population is considered adequate, but subpopulations remain at risk for iodine deficiency and a biomarker of individual iodine status has yet to be determined. The purpose of this study was to determine whether a 3 day titration diet, providing known quantities of iodized salt, is reflected in 24 h urinary iodine concentration (UIC), serum iodine, and thyroglobulin (Tg). Methods: A total of 10 participants (31.3 ± 4.0 years, 76.1 ± 6.3 kg) completed three, 3 day iodine titration diets (minimal iodine, US RDA, (United States Recommended Daily Allowance), and 3× RDA). The 24 h UIC, serum iodine, and Tg were measured following each diet. The 24 h UIC and an iodine-specific food frequency questionnaire (FFQ) were completed at baseline. Results: UIC increased an average of 19.3 μg/L for every gram of iodized salt consumed and was different from minimal to RDA (p = 0.001) and RDA to 3× RDA diets (p = 0.04). Serum iodine was different from RDA to 3× RDA (p = 0.006) whereas Tg was not responsive to diet. Baseline UIC was associated with iodine intake from milk (r = 0.688, p = 0.028) and fish/seafood (r = 0.646, p = 0.043). Conclusion: These results suggest that 24 h UIC and serum iodine may be reflective of individual iodine status and may serve as biomarkers of iodine status.