Two different methods for measuring the bending stiffness of lipid membranes are used and further developed: fluctuation analysis and vesicle electrodeformation. For this purpose, fast camera imaging was employed minimizing the experimental effort. The methods were applied to study the effect of cholesterol on the bending stiffness of two types of membranes. We explored giant vesicles prepared from dioleoylphosphatidylcholine–cholesterol and sphingomyelin–cholesterol mixtures. The results show that the effect of cholesterol on the bending stiffness is quite different and lipid-specific. While the bending stiffness of dioleoylphosphatidylcholine membranes does not change significantly, sphingomyelin membranes become more flexible with the addition of cholesterol. Finally, we report data on vesicles prepared from lipid extracts of the plasma membrane of human red blood cells and investigate the influence of naturally present transmembrane peptides. The latter molecules do not alter the membrane stiffness significantly.