Walled cells, such as plants, fungi, and bacteria cells, possess a high internal hydrostatic pressure, termed turgor pressure, that drives volume growth and contributes to cell shape determination. Rigorous measurement of turgor pressure, however, remains challenging, and reliable quantitative measurements, even in budding yeast are still lacking. Here, we present a simple and robust experimental approach to access turgor pressure in yeasts based upon the determination of isotonic concentration using protoplasts as osmometers. We propose three methods to identify the isotonic condition – 3D cell volume, cytoplasmic fluorophore intensity, and mobility of a cytGEMs nano-rheology probe – that all yield consistent values. Our results provide turgor pressure estimates of 1.0 ± 0.1 MPa forS. pombe, 0.49 ± 0.01 MPa forS. japonicus, 0.5 ± 0.1 MPa forS. cerevisiae W303aand 0.31 ± 0.03 MPa forS. cerevisiae BY4741. Large differences in turgor pressure and nano-rheology measurements between theS. cerevisiaestrains demonstrate how fundamental biophysical parameters can vary even among wildtype strains of the same species. These side-by-side measurements of turgor pressure in multiple yeast species provide critical values for quantitative studies on cellular mechanics and comparative evolution.