The objective of this invention is to create a double thyA folA knockout Escherichia coli (E. coli) strain for antifolate screening against DHFR of malaria and other parasites. This strain is used together with a plasmid expressing DHFR-TS from the desired pathogenic organism, which constitutes an anti-DHFR assay against the pathogenic organism of interest. The benefit of this invention is that there is no interference from either host DHFR or trimethoprim, a bacterial DHFR inhibitor. This tool is easy to use and maintain. It provides quick and reliable results as compared with conventional anti-malarial and anti-parasitic assays. This invention should facilitate discovery of new anti-DHFR compounds against malaria and other parasitic diseases.