Multiplex assays are a powerful tool for molecular subtyping of pathogens, which is crucial for rapid diagnosis, surveillance and identification and containment of outbreaks. The Luminex xMAP technology allows detection of up to 500 different analytes per sample in a high-throughput format through a liquid bead suspension array. Different types of Luminex assays can be envisaged. Each assay has its advantages and limitations, some of which are related to the inherent design of the primers and probes used. Software that predicts melting temperatures, hybridization structures and specificity for different types of oligonucleotides, and thereby simulating the multiplex assays in silico can facilitate the design of multiplex assays by reducing time and cost of experimentation.We have compared two commercial software packages for the development and in silico simulation of a ligation dependent amplification (LDA) assay and a direct hybridization assay for the subtyping of a pathogen. The main findings, limitations and challenges will be discussed.