Although the plaque reduction neutralization test (PRNT) is considered the reference for measuring neutralizing antibodies for Yellow fever virus (YFV), the micro-focus reduction neutralization test (mFRNT) presents many advantages including a higher throughput, a reduced incubation time, and allows the automatic foci count. So, here we describe a detailed mFRNT method that provides a faster neutralization test essential for non-clinical and clinical studies and vaccine design.