Since the global outbreak of Mpox in 2022, interest in seroepidemiological and vaccine studies has grown rapidly. One aspect of interest in these studies is the detection of neutralizing antibodies to the causative agent, monkeypox virus (MPXV). Determination of neutralizing antibodies using a (micro)-neutralization test (NT) is the gold standard method, which is time consuming and labor intensive. Here, we present the comparison of different methods to facilitate MPXV NT analysis resulting in the establishment of an optimized MPXV NT protocol. The optimized protocol is based on a recombinant eGFP-expressing MPXV and a recombinant mCherry-expressing VeroE6 cell line. Using the so-called Xmas-NT, the incubation time can be reduced by 60%. In addition, compared to the conventional NT, the documentation time of the Xmas-NT is significantly reduced by 80%, making it suitable for serological high throughput screening studies.