The iodine status of populations is conventionally assessed using spot urinary samples to obtain a median urinary iodine concentration (UIC) value, which is assessed against standard reference cut-offs. The assumption that spot UIC reflects daily iodine intake may be flawed because of high day-to-day variability and variable urinary volume outputs. This study aimed to compare iodine status in a sample of South African adults when determined by different approaches using a spot urine sample (median UIC (MUIC), predicted 24 h urinary iodine excretion (PrUIE) using different prediction equations) against measured 24 h urinary iodine excretion (mUIE). Both 24 h and spot urine samples were collected in a subsample of participants (n = 457; median age 55 year; range 18–90 year) in theWorld Health Organization Study on global AGEing and adult health (SAGE) Wave 2 in South Africa, in 2015. Kawasaki, Tanaka, and Mage equations were applied to assess PrUIE from predicted urinary creatinine (PrCr) and spot UIC values. Adequacy of iodine intake was assessed by comparing PrUIE and mUIE to the Estimated Average Requirement of 95 μg/day, while the MUIC cut-off was <100 >μg/L. Bland Altman plots assessed the level of agreement between measured and predicted UIE. Median UIC (130 μg/L) indicated iodine sufficiency. The prediction equations had unacceptable bias for PrUIE compared to measured UIE. In a sample of adult South Africans, the use of spot UIC, presented as a group median value (MUIC) provided similar estimates of inadequate iodine status, overall, when compared to EAR assessed using measured 24 h iodine excretion (mUIE). Continued use of MUIC as a biomarker to assess the adequacy of population iodine intake appears warranted.