In vitro determination of hemolytic properties is a common and important method for preliminary evaluation of cytotoxicity of chemicals, drugs, or any blood-contacting medical device or material. The method itself is relatively straightforward, however, protocols used in the literature vary substantially. This leads to significant difficulties both in interpreting and in comparing the obtained values. Here, we examine how the different variables used under different experimental setups may affect the outcome of this assay. We find that certain key parameters affect the hemolysis measurements in a critical manner. The hemolytic effect of compounds tested here varied up to fourfold depending on the species of the blood source. The use of different types of detergents used for generating positive control samples (i.e., 100% hemolysis) produced up to 2.7-fold differences in the calculated hemolysis ratios. Furthermore, we find an expected, but substantial, increase in the number of hemolyzed erythrocytes with increasing erythrocyte concentration and with prolonged incubation time, which in turn affects the calculated hemolysis ratios. Based on our findings we propose an optimized protocol in an attempt to standardize future hemolysis studies.