Lycopene, beta-carotene, coenzyme Q10, and lutein are minor constituents of palm oil that are removed during biodiesel production to produce light-coloured oils. With the aim to investigate the recovery of these valuable compounds, a separation method was developed to quantify carotenoids and coenzyme Q10 in palm oil by ultra-high-performance supercritical fluid chromatography. Due to the presence of interferents, different clean-up procedures were evaluated; however, these approaches were ineffective and the separation method was developed without this step. The chemometric method multivariate curve resolution-alternating least squares was employed to properly quantify lycopene, beta-carotene, and coenzyme Q10 in the presence of interferents. Lutein was sufficiently resolved to be quantified by a univariate method. Lycopene concentration was below the limit of quantification 3.12 μg/mL (3.12 × 10−3 kg/m3). Beta-carotene concentration was determined as being 183.48 ± 1.66 μg/mL (183.48 ± 1.66 × 10−3 kg/m3). Coenzyme Q10 concentration was lower than the limit of detection 4.22 μg/mL (4.22 × 10−3 kg/m3) and lutein concentration 9.24 μg/mL (9.24 × 10−3 kg/m3) was below the limit of quantification. The study showed the analytical challenges associated with the separation and quantification of minor constituents of a highly complex matrix such as palm oil and demonstrated that the recovery of beta-carotene could be economically viable due to its wide range of application in industry.