Background: Tomatoes (Solanum lycopersicum) are an economically and nutritionally important crop colored by carotenoids such as lycopene and β-carotene. Market diversification and interest in the health benefits of carotenoids has created the desire in plant, food, and nutritional scientists for improved extraction and quantification protocols that avoid the analytical bottlenecks caused by current methods. Objective: Our objective was to compare standard and rapid extraction as well as chromatographic separation methods for tomato carotenoids. Method: Comparison was based on accuracy and the ability to discriminate between alleles and genetic backgrounds. Estimates of the contribution to variance in the presence of genetic and environmental effects were further used for comparison. Selections of cherry and processing tomatoes with varying carotenoid profiles were assessed using both established extraction and HPLC-diode array detector (HPLC-DAD) methods and rapid extraction and ultra-HPLC-DAD (UHPLC-DAD) protocols. Results: Discrimination of alleles in samples extracted rapidly (<5 min/sample) was similar to samples extracted using a standard method (10 min/sample), although carotenoid concentrations were lower due to reduced extraction efficiency. Quantification by HPLC-DAD (21.5 min/sample) and UHPLC-DAD (4.2 min/sample) were comparable, but the UHPLC-DAD method could not separate all carotenoids and isomers of tangerine tomatoes. Random effects modeling indicated that extraction and chromatographic methods explained a small proportion of variance compared with genetic and environmental sources. Conclusions: The rapid extraction and UHPLC-DAD methods could enhance throughput for some applications compared with standard protocols.