Efficient extraction of total proteins from soil microorganisms is tedious because of small quantity. In this regard, an improved method for extraction of whole cell proteins is developed from soil microorganisms, Saccharomyces cerevisiae and Pichia pastoris. of which the cell wall are very strong. Pretreatment with NH4OH prior to the final extraction using NaOH/SDS was tried under the basis that ammonium ion was possible to enhance the permeability and/or to weaken the yeast cell walls. The pre-treatment of yeast cells with NH4OH drastically enhanced the protein extraction when it was compared with control (without NH4OH pre-treatment). At the pre-treatment of 0.04 N NH4OH at pH 9.0, about 3 fold of proteins was obtained from p. pastoris. Ammonium hydroxide appears to penetrate into the yeast cell walls more readily at basic pH. The effect of NH4OH pretreatment was pH dependent. The methods developed in this experiment might be applicable for an effective extraction of yeast proteins for the purpose of biochemical studies, especially proteomic analysis.