Non-conventional yeasts (NCYs), i.e. all yeasts other than Saccharomyces cerevisiae, are emerging as novel production strains and gain more and more attention to exploit their unique properties. Yet, these yeasts can hardly compete against the advanced methodology and genetic tool kit available for exploiting and engineering S. cerevisiae. Currently, for many NCYs one has to start from scratch to initiate molecular genetic manipulations, which is often time consuming and not straight-forward. More so because utilization of S. cerevisiae tools based on short-flank mediated homologous recombination or plasmid biology are not readily applicable in NCYs. Here we present a script with discrete steps that will lead to the development of a basic and expandable molecular toolkit for ascomycetous NCYs and will allow genetic engineering of novel platform strains. For toolkit development the highly efficient in vivo recombination efficiency of S. cerevisiae is utilized in the generation and initial testing of tools. The basic toolkit includes promoters, reporter genes, selectable markers based on dominant antibiotic resistance genes and the generation of long-flanking homology disruption cassettes. The advantage of having pretested molecular tools that function in a heterologous host facilitate NCY strain manipulations. We demonstrate the usefulness of this script on Saccharomycopsis schoenii, a predator yeast with useful properties in fermentation and fungal biocontrol.