Fruits and vegetables are the major sources of biologically active compounds, and carotenoids and tocopherols constitute important groups in human diets. Bioavailability is a critical feature in the assessment of the role of micronutrients in human health, and the approaches to this issue include in vitro and in vivo methods. Our aim was to evaluate the bioavailability of carotenoids and tocopherols present in broccoli and to compare in vitro and in vivo approaches. Fourteen apparently healthy volunteers consumed 200 g broccoli once a day for seven days. Blood samples were drawn at baseline and after intervention to determine changes in lutein, β-carotene, and Α- and γ-tocopherol as relevant phytochemicals provided with this vegetable. Broccoli also was subjected to simulated gastrointestinal digestion to assess changes related to preabsorptive processes. Analytes in serum and at each phase of the digestion were assayed by high-performance liquid chromatography. During the intervention, the amounts supplied dally ranged from 2.4 to 3.1 mg lutein, 1.4 to 1.8 mg β-carotene, 4.5 to 6.8 mg Α-tocopherol, and 0.8 to 1.8 mg γ-tocopherol. Significant changes in serum in both men and women were observed only for lutein, whereas for γ-tocopherol a significant change was detected in women. No changes were observed for Α-tocopherol, β-carotene, retinol, the Α-tocopherol-to-cholesterol ratio, or serum lipids. Using the in vitro model, more than 75% of lutein, β-carotene, γ-tocopherol, and Α-tocopherol remained at the duodenal phase, whereas Incorporation Into the supernatants accounted for <20% of the initial content in food. Regular consumption of broccoli at dietary levels increased serum concentrations of lutein and γ-tocopherol without affecting Α-tocopherol or β-carotene status in serum. The behavior of these phytochemicals under in vitro gastrointestinal conditions does not fully explain the changes observed in vivo.