New technologies are promising for the use of short-term instruments for dietary data collection; however, innovative tools should be validated against objective biomarkers. The aim of the present study was to investigate the validity of a Web-based, self-administered dietary record (DR) tool using protein, K and Na intakes against 24 h urinary biomarkers (24 h U). Of the total participants, 199 adult volunteers (104 men and 95 women, mean age 50·5 (23-83 years)) of the NutriNet-Santé Study were included in the protocol. They completed three non-consecutive-day DR and two 24 h U on the first and third DR days. Relative differences between reported (DR) and measured (24 h U) intakes were calculated from the log ratio (DR/24 h U) for protein, K and Na intakes: -14·4,+2·6 and -2·1 % for men; and -13·9, -3·7 and -8·3 % for women, respectively. The correlations between reported and true intakes were 0·61, 0·78 and 0·47 for men and 0·64, 0·42 and 0·37 for women for protein, K and Na, respectively. Attenuation factors, that represent attenuation of the true diet-disease relationship due to measurement error (a value closer to 1 indicating lower attenuation), ranged from 0·23 (Na, women) to 0·60 (K, men). We showed that the Web-based DR tool used in the NutriNet-Santé cohort study performs well in estimating protein and K intakes and fairly well in estimating Na intake. Furthermore, three non-consecutive-day DR appear to be valid for estimating usual intakes of protein and K, although caution is advised regarding the generalisability of these findings to other nutrients and general population.