The study generally aimed to evaluate a Loop-mediated Isothermal Amplification (LAMP) technique in amplifying the gene expressing the ESBL of E. coli. Specifically, it aimed to optimize the protocol of LAMP assay for the detection ESBL-producing Escherichia coli and to compare diagnostic validity and level of agreement with PCR. A total of 106 DNA samples were used in the study. For the optimization of LAMP protocol, 12 tubes with ESBL E. coli and 12 tubes with nuclease-free water were incubated at a thermal cycler in temperatures ranging from 60-63ºC for 30-90 minutes. Validation of results was based on the ability of the positive controls to change in color by adding a SYBR dye, fluorescence under ultraviolet light and the presence of marker in gel electrophoresis upon query. The agreement using Kappa statistics between LAMP and PCR was used to determine the level of agreement of the two methods. For LAMP assay amplification, amplification of the target gene was observed at 63ºC for 60 minutes while the Kappa coefficient was only 0.205, which means that there was a slight agreement between the LAMP and PCR.