The non-osmotic volume (NOV) of Polypodium vulgare guard cells (GC) was calculated by measuring the volume of protoplasts immersed in three different concentrations of manitol. Before protoplasts could be measured, they had to be obtained in sufficient numbers. GC protoplasts were obtained by incubating epidermal strips in enzyme solution for an average of six hours. Sufficient numbers of protoplasts were obtained on half the attempted trial days. It was found that only very young leaves from the growth room were able to be successfully digested. Even very young leaves grown outside were resistant to the enzyme solution. Cutting up the epidermis into fine strips with a razor prior to incubation and periodically knocking the Petri dish on the table during incubation both helped to increase the yield of GC protoplasts. The guard cell protoplasts were not separated from the rest of the epidermal debris, since they could be easily distinguished from the epidermal and mesophyl protoplasts based on size, colour and chloroplast concentration. Once sufficient data had been obtained, regression analysis using the Boyle van’t Hoff equation was performed in order to determine the NOV of the GCs. The measurements of protoplast volume at the 200mM level displayed a lot of variation. The standard deviation from the mean was larger in the 200mM concentration solution in almost every trial. This was likely due to the swelling and bursting of the protoplasts at this concentration. With the 200mM level included, it was found that the guard cells had an NOV between 126.52µm3 and 1423.8µm3 with an average of 303.98µm3. Omitting the 200mM level from the calculations yielded less variable and more plausible values for NOV between 135.9µm3 and 3359.9µm3 with an average of 2039µm3. Epidermal strips were exposed to fusicoccin to discover whether or not the GCs were active. Separate batches of epidermal strips were incubated in ion solutions of KCl and NaCl of many different concentrations. Each batch was split in two with one half incubated in the dark and the other in the light. In all cases, stomata failed to open. Proton pumping is the first step in the mechanism of stomatal opening. Fusicoccin is a well known activator of the ATP powered proton pump so the fact that the stomata remained closed in all cases was a surprising result.