We describe herein a method for quantifying and ranking the puncture resistance of lithium-ion battery separators, which can be used as a quality-control tool for determining the in-plane uniformity of their mechanical properties. Mechanical properties were probed over length scales comparable to the size of electrode active material particles. We determined the elastic modulus and hardness of four polymeric and four organic–inorganic composite separators through nanoindentation, and examined their morphology with scanning electron microscopy. Our results show that ceramic-enhanced separators can have improved mechanical properties compared to polymeric separators (3× increase in elastic modulus, 2× increase in hardness). Composite separators made of ultra-high molecular weight polyethylene and fumed alumina displayed the highest hardness. The results also indicate that the mechanical properties of ceramic-enhanced separators depend strongly on materials choices and processing conditions (in particular, their thermal history). Furthermore, inclusion of ceramics into a separator does not guarantee improved mechanical properties.