Combination of the pYeDP60 yeast expression system with a modified version of the improved uracil-excision (USER) cloning technique provides a new powerful tool for high-throughput expression of eukaryotic cytochrome P450s. The vector presented is designed to obtain an optimal 5' untranslated sequence region for yeast (Kozak consensus sequence), and has been tested to produce active P450s and NADPH-cytochrome P450 oxidoreductase (CPR) after 5' end silent codon optimization of the cDNA sequences. Expression of two plant cytochrome P450s, Sorghum bicolor CYP79A1 and CYP71E1, and S. bicolor CPR2 using the modified pYeDP60 vector in all three cases produced high amounts of active protein. High-throughput functional expression of cytochrome P450s have long been a troublesome task due to the workload involved in cloning of each individual P450 into a suitable expression vector. The redesigned yeast P450 expression vector (pYeDP60u) offers major improvements in cloning efficiency, speed, fidelity, and simplicity. The modified version of the USER cloning system provides great potential for further development of other yeast vectors, transforming these into powerful high-throughput expression vectors.