The selection of cultivars with high carotenoid content represents a novel goal in the genetic improvement of oilseed rape. The successful development of an efficient method for the quantification of carotenoids in oilseed rape is a prerequisite for this breeding initiative. In the present study, ultrasound-assisted extraction was combined with a spectrophotometric method to quantify seed carotenoid levels in oilseed rape. The key variables were optimised as follows: the solvent used was a 1 : 1 (v : v) mixture of petroleum ether and acetone, the solid to solvent concentration was 0.08 g/mL, the ultrasound frequency and power were set at 45/100 (kHZ/w), and three extractions of 15 min each were performed. Compared with the conventional solvent extraction method, the time required for extraction using the ultrasound-assisted extraction method was significantly shortened (by 24 times), and the reproducibility was also remarkably improved. These results indicate that ultrasound-assisted spectrophotometric quantification is a rapid, accurate, simple and low cost tool for the screening and selection of rapeseed cultivars with high carotenoid content.