Abstract: The extraction of lutein and β-carotene from spinach (Spinacia oleracea L.) leaves is important to the dietary supplement industry. A Box-Behnken design and response surface methodology (RSM) were used to investigate the effect of process variables on the ultrasound-assisted extraction (UAE) of lutein and β-carotene from spinach. Three independent variables, extraction temperature (°C), extraction power (%) and extraction time (min) were studied. Thin-layer chromatography (TLC) followed by UV visualization and densitometry was used as a simple and rapid method for both identification and quantification of lutein and β-carotene during UAE. Methanol extracts of leaves from spinach and authentic standards of lutein and β-carotene were separated by normal-phase TLC with ethyl acetate-acetone (5:4 (v/v)) as the mobile phase. In this study, the combination of TLC, densitometry, and Box–Behnken with RSM methods were effective for the quantitative analysis of lutein and β-carotene from spinach extracts. The resulting quadratic polynomial models for optimizing lutein and β-carotene from spinach had high coefficients of determination of 0.96 and 0.94, respectively. The optimal UAE settings for output of lutein and β-carotene simultaneously from spinach extracts were an extraction temperature of 40 °C, extraction power of 40% (28 W/cm3) and extraction time of 16 min. The identity and purity of each TLC spot was measured using time-of-flight mass spectrometry. Therefore, UAE assisted extraction of carotenes from spinach can provide a source of lutein and β-carotene for the dietary supplement industry