Ca²⁺ sparks are the elementary units of Ca²⁺ signaling in striated muscle fibers that appear as highly localized Ca²⁺ release events through ryanodine receptor (RyR) Ca²⁺ release channels in the sarcoplasmic reticulum (SR). While these events are commonly observed in resting cardiac myocytes, they are rarely seen in resting skeletal muscle fibers. Since Ca²⁺ spark analysis can provide extensive data on the Ca²⁺ handling characteritsics of normal and diseased striated muscle, there has been interest in developing methods for observing Ca²⁺ sparks in skeletal muscle. Previously, we discovered that stress generated by osmotic pressure changes induces a robust Ca²⁺ spark response confined in close spatial proximity to the sarcolemmal membrane in wild-type intact mammalian muscles. Our studies showed these peripheral Ca²⁺ sparks (PCS) were altered in dystrophic or aged skeletal muscles. Other methods to induce Ca²⁺ sparks include permeabilization of the sarcolemmal membrane with detergents, such as saponin. In this chapter, we will discuss the methods for isolation of muscle fibers, the techniques for inducing Ca²⁺ sparks in these isolated fibers, and provide guidance on the analysis of data from these experiments.