Dietary validation studies of self-reported fruit and vegetable intake should ideally include measurement of plasma biomarkers of intake. The aim was to conduct a validation study of self-reported fruit and vegetable intakes in adults, using the Australian Eating Survey (AES) food frequency questionnaire (FFQ), against a range of plasma carotenoids. Dietary intakes were assessed using the semi-quantitative 120 item AES FFQ. Fasting plasma carotenoids (α- and β-carotene, lutein/zeaxanthin, lycopene and cryptoxanthin) were assessed using high performance liquid chromatography in a sample of 38 adult volunteers (66% female). Significant positive correlations were found between FFQ and plasma carotenoids for α-carotene, β-carotene and lutein/zeaxanthin (52%, 47%, 26%, p < 0.001, 0.003, 0.041; respectively) and relationships between plasma carotenoids (except lycopene) and weight status metrics (BMI, waist circumference, fat mass) were negative and highly significant. The results of the current study demonstrate that carotenoid intakes as assessed by the AES FFQ are significantly related to plasma concentrations of α-carotene, β-carotene and lutein/zeaxanthin, the carotenoids commonly found in fruit and vegetables. Lower levels of all plasma carotenoids, except lycopene, were found in individuals with higher BMI. We conclude that the AES can be used to measure fruit and vegetable intakes with confidence.