Introduction: Urinary biomarkers are invaluable for non-invasive assessment of nutrient status but urine collection can be cumbersome, especially in infants. Cotton fiber-based sanitary napkins offer a practical and cost-effective solution for urine collection, but could potentially interact with the urine and affect biomarker measurements. In this study, we aimed to validate whether urine extracted from sanitary napkins, can be used for urinary iodine and methylmalonic acid (MMA) analysis. Methods: We collected urine samples from volunteers following different dietary patterns (n = 30). Daily intake of iodine and vitamin B12 were estimated by food frequency questionnaire and supplement use was recorded. Samples were evaluated both in their native state and after adding them on sanitary napkins, with subsequent re-collection. Urinary iodine was analyzed by ICP-MS, MMA by LC-MS/MS and creatinine photometrically. To compare the agreement of measurement between native and processed samples, intraclass correlation coefficients (ICC) were calculated. Results: Daily estimated iodine intake varied from 57.8–216.5 µg/day and vitamin B12 from 1.2–685 µg/day yielding a broad range of urinary iodine: (5.60–151.3 µg/L) and MMA (0.70–2.37 mg/g creatinine). Native and processed urine showed an excellent agreement of measurement for iodine (ICC = 1.0), MMA (ICC = 0.99), creatinine (ICC = 0.99) as well as the ratio of MMA/ creatine (ICC = 0.95) with p < 0.001 for all, indicating no interaction between analytes and napkin. Conclusion: We demonstrate the validity of urine collection using sanitary napkins to measure urinary iodine, MMA and creatinine over a broad range of concentrations. Our approach is affordable and straightforward, and could streamline urine collection in large-scale studies.