Resumo. Abstract: We optimized a methodology for plankton environmental DNA detection of the invasive golden mussel and validated it in samples from a Southern Brazil reservoir. Limnoperna fortunei is a successful invasive alien species that causes significant impacts on freshwater ecosystems. We adjusted and validated the methodology to detect L. fortunei in plankton samples, with a SYBR Green assay. Based on the standard curve analysis, the observed theoretical minimal qPCR detection level was 0.0005625 ng.µL-1 (R2 = 0.99) at a PCR quantification cycle of 14.09–29.56. We also presented a practical guide to be used in monitoring and detection of L. fortunei. The optimized protocol was efficient in detecting L. fortunei and can be used to monitor already infested environments or invasions in new environments.