Wheat is one of the major staple crops around the world. A transient expression system is crucial for gene functional studies in wheat as stable transfection is still difficult in most cultivars. Protoplasts could serve as a versatile transient expression tool in wheat research. Here, we describe protocols for wheat protoplast isolation and transfection that are enabled by cellulase R-10 and macerozyme R-10 containing enzymatic solution and polyethylene glycol-mediated method, respectively. In addition, we show an example of efficiency evaluation of the emerging base editors in wheat protoplasts. These protocols are of wide use in both conventional gene functional analysis and reagent functionality evaluation of genome editing in wheat.