The preparation and observations of spheroplast W303 cells are described with Environmental Scanning Electron Microscope (ESEM). The spheroplasting conversion was successfully confirmed qualitatively, by the evaluation of the morphological change between the normal W303 cells and the spheroplast W303 cells, and quantitatively, by determining the spheroplast conversion percentage based on the OD800 absorbance data. From the optical microscope observations as expected, the normal cells had an oval shape whereas spheroplast cells resemble a spherical shape. This was also confirmed under four different mediums, that is, yeast peptone-dextrose (YPD), sterile water, sorbitol-EDTA-sodium citrate buffer (SCE), and sorbitol-Tris-Hcl-CaCl2 (CaS). It was also observed that the SCE and CaS mediums had a higher number of spheroplast cells as compared to the YPD and sterile water mediums. The OD800 absorbance data also showed that the whole W303 cells were fully converted to the spheroplast cells after about 15 minutes. The observations of the normal and the spheroplast W303 cells were then performed under an environmental scanning electron microscope (ESEM). The normal cells showed a smooth cell surface whereas the spheroplast cells had a bleb-like surface after the loss of its integrity when removing the cell wall.