Flowering trees are considered the producers of allergenic agent carried by pollen protein. The isolation of protein of interest from intracellular compartment into a solution of well-defined composition especially from never isolated species involves the manipulation of several parameters. The aim of this work is to find the best isolation method for pollen protein of the widely distributed flowering fruit species in Malaysia includes Mangifera indica, Mangifera odorata, Durio graviolens, Durio zibethinus, Syzygium aqueum (red flower), Syzygium aqueum (white flower), to give the maximum yield extract of crude protein. The most efficient buffer to be used specifically for each species were as followed; Mangifera indica (Tris HCl 0.5M pH 6.8 with 1.3 mg/ml); Mangifera odorata (Tris HCl 0.5M pH 6.8 with 1.8 mg/ml); Durio graviolens (PBS 0.02M pH 7.4 with 1.6 mg/ml); Durio zibethinus (PBS 0.02M pH 7.4 with 2.2 mg/ml); Syzygium aqueum -red flower (Tris HCl 0.5M pH 6.8 with 1.0 mg/ml); Syzygium aqueum-white flower (PBS 0.02M pH 6.8 with 1.7 mg/ml). Pectinase activity reveals the best ammonium sulfate percentage for Mangifera indica, Mangifera odorata, Durio graviolens, Durio zibethinus, Syzygium aqueum -red flower, Syzygium aqueum-white flower is either 80% or 85%. Different types of buffer definitely have different ability on the protein solubilization, while the 80% of salt precipitation was the most ideal percentage for salting out the protein in every sampled species.