Plant carotenoids, the precursors of vitamin A display several important biological functions as antioxidants and anti-carcinogens. The oilseed crops, owing to their high oil content, form a good matrix for the bioavailability of β-carotene, thereby providing potential targets for biofortification to combat vitamin A deficiency (VAD). However, the screening and characterization of these crops, that otherwise contain very low levels of pro-vitamin A carotenoids has been difficult owing to their poor recovery and strong binding to the oil matrix. Here, we report a rapid method for high volume HPLC analysis involving the extraction and determination of β-carotene in four oilseed crops (peanut, soybean, sunflower and mustard). This included a comprehensive study of the factors that potentially influence the qualitative and quantitative yields of β-carotene in these crops. This is the first crop-independent HPLC method for the quantification of pro-vitamin A carotenoids that shows excellent recovery and reproducibility (>90 percentage recovery in oil) using small tissue sample and is capable of processing up to 30 samples per day. The protocol is sensitive, and enables better detection and separation of individual carotenoids by reducing artefacts during extraction, purification and chromatography that can be used for routine screening of oilseeds.