Reliable tools to evaluate diet are needed, particularly in life periods such as adolescence in which a rapid rate of growth and development occurs. We assessed the biochemical validity of a self-administered food frequency questionnaire (FFQ) in a sample of Spanish male adolescents using carotenoids and vitamin E and D data. We analyzed data from 122 male adolescents aged 15–17 years of the INMA-Granada birth cohort study. Adolescents answered a 104-item FFQ and provided a non-fasting blood sample. Mean daily nutrient intakes and serum concentration were estimated for main carotenoids (lutein-zeaxanthin, β-cryptoxanthin, lycopene, α-carotene and β-carotene), vitamins E and D and also for fruit and vegetable intake. Pearson correlation coefficients (r) and the percentage of agreement (same or adjacent quintiles) between serum vitamin concentrations and energy-adjusted intakes were estimated. Statistically significant correlation coefficients were observed for the total carotenoids (r = 0.40) and specific carotenoids, with the highest correlation observed for lutein–zeaxanthin (r = 0.42) and the lowest for β-carotene (0.23). The correlation coefficient between fruit and vegetable intake and serum carotenoids was 0.29 (higher for vegetable intake, r = 0.33 than for fruit intake, r = 0.19). Low correlations were observed for vitamin E and D. The average percentage of agreement for carotenoids was 55.8%, and lower for vitamin E and D (50% and 41%, respectively). The FFQ may be an acceptable tool for dietary assessment among male adolescents in Spain.