The performance and the reliability of a SYBR Green I fluorescence-based assay to assess drug susceptibility in routine monitoring were evaluated in 138 Plasmodium falciparum clinical samples. Blood samples were studied for susceptibility to four antimalarial drugs by the SYBR Green I based assay, with the traditional [(3)H]-hypoxanthine isotopic assay as a reference. The two methods were observed to have similar geometric means of IC50s and IC90s, and high correlation (r=0.93 for IC50s and r=0.94 for IC90s) for the drugs tested. The strength of agreement estimated by using concordance coefficient correlation was from almost perfect to substantial for IC50s. Our data demonstrate (i) the reliability of a simple, rapid and easy to use fluorescence-based assay for the routine monitoring of susceptibility of P. falciparum clinical isolates, and (ii) the possible switch from the traditional in vitro drug sensitivity assay to the SYBR Green I method, because previous data acquired by the isotopic assay were comparable with those obtained by the SYBR Green I method. We conclude that this assay will provide an easier method for testing drug susceptibility of malaria parasites, especially in malaria-endemic countries, where there is massive implementation of new artemisinin-based combination therapies.