Production of carotenoids and lipids by Rhodotorula toruloides CBS 14 cultivated on wheat straw hydrolysate was investigated. An ultra-high-performance liquid chromatography (UHPLC) method for carotenoid quantification was developed and validated. Saponification effects on individual carotenoid quantification were identified, and lipid and carotenoid kinetics during cultivation were determined. The carotenoids β-carotene, γ-carotene, torularhodin, and torulene were identified; β-carotene was the major carotenoid, reaching a maximum of 1.48 mg/100 g dry weight. Recoveries of the carotenoids were between 66% and 76%, except torulene and torularhodin, which had lower recoveries due to saponification effects. Total carotenoid content in saponified and unsaponified yeast extract, respectively, determined by UHPLC or photometer, respectively, was 1.99 mg/100 g and 4.02 mg β-EQ/100 g dry weight. Growth kinetics showed a positive correlation between carotenoid content and lipid accumulation. β-carotene was the major carotenoid at all time points. At the end of the cultivation, triacylglycerols (TAGs) were the major lipid class, with 58.1% ± 3.32% of total lipids. There was also a high proportion of free fatty acids, reaching from 20.5% to 41.8% of total lipids. Oleic acid (C18:1) was the major fatty acid. The lipid yield at the end of the cultivation was 0.13 g/g of sugar consumed.