Dark green leafy vegetables are primary food sources for lutein and β-carotene, however these bioactives have low bioavailability. The effects of mechanical and thermal processing as well as fat addition and fat type on lutein and β-carotene liberation and in vitro accessibility from spinach were investigated. Lutein liberation and in vitro accessibility were three-fold higher from spinach puree compared to whole leaves. Results for β-carotene liberation were similar, whereas that of β-carotene accessibility was only about two-fold. Steaming had no or a negative effect on carotenoid liberation. Fat addition increased β-carotene liberation from raw and steamed puree, but reduced lutein liberation from steamed leaves and raw puree. Fat types affected β-carotene differently. Butter addition led to a 2.5 fold increased liberation from raw spinach puree, while the effect of olive and peanut oil was significantly lower, but only minor effects were observed for lutein.