The objective of present study was to optimize loop mediated isothermal amplification (LAMP) assay for the diagnosis of Babesia felis in cats. LAMP primers were designed recognizing four sections of 18SribosomalRNA (18S rRNA) gene of B. felis. The blood samples of cats microscopically positive for Babesia felis were further used to extract deoxyribo neuclic acid (DNA) and the reaction mixture of 25 µL was standardized at 63°C temperature for 1 hour. LAMP assay provided more positive samples than conventional polymerase chain reaction (PCR). The prevalence of B. felis was also determined in cats using this optimized LAMP assay and it was found that the prevalence was more in younger cats as compare to adults. The application of LAMP can be helpful in rapid, reliable and cost effective diagnosis of B. felis in field.