Identification and characterization of key enzymes associated with cell wall biosynthesis and modification is fundamental to gain insights into cell wall dynamics. However, it is a challenge that activity assays of glycosyltransferases are very low throughput and acceptor substrates are generally not available. We optimized and validated microscale thermophoresis (MST) to achieve high throughput screening for glycosyltransferase substrates. MST is a powerful method for the quantitative analysis of protein–ligand interactions with low sample consumption. The technique is based on the motion of molecules along local temperature gradients, measured by fluorescence changes. We expressed glycosyltransferases as YFP-fusion proteins in tobacco and optimized the MST method to allow the determination of substrate binding affinity without purification of the target protein from the cell lysate. The application of this MST method to the β-1,4-galactosyltransferase AtGALS1 validated the capability to screen both nucleotide-sugar donor substrates and acceptor substrates. We also expanded the application to members of glycosyltransferase family GT61 in sorghum for substrate screening and function prediction. This method is rapid and sensitive to allow determination of both donor and acceptor substrates of glycosyltransferases. MST enables high throughput screening of glycosyltransferases for likely substrates, which will narrow down their in vivo function and help to select candidates for further studies. Additionally, this method gives insight into biochemical mechanism of glycosyltransferase function.