Stevia rebaudiana Bertoni is a perennial shrub from South America that produces steviol glycosides which are 200-300 times sweeter than sugar. Stevioside and rebaudioside A are the main sweetening components of its leaves. Steviol glycosides are diterpenoids whose biosynthetic pathways have four steps in common with gibberellic acid formation. The most important enzyme in the biosynthetic pathway expressed by the gene UGT76G1 is referred to as UDP-glycosyltransferase 76G1. It converts stevioside into rebaudioside A. The former has a bitter aftertaste and is a poorer sweetener but is most abundant. This enzyme can be produced in a next generation recombinant way by Escherichia coli and Saccharomyces cerevisiae. Trichoderma longibrachiatum produces the enzyme β-1,3-glucanase enzyme, which can perform a transglycosylation between stevioside to gain rebaudioside A. In our study, a full-factorial statistical experimental design that applies different glycosyl donors, temperatures, enzyme-to-substrate ratios and pH's as factors in order to achieve higher Reb A ratios in S. rebaudiana extracts after transglycosylation is reported. The presented statistical design was appropriate to indicate relevant and significant factors, providing a good basis for an upcoming experimental design of a real-world optimization.