Autophagy is an important intracellular degradation system which is implicated in many physiological and pathological processes. During autophagy, cytosolic constituents such as organelles and macromolecules are engulfed by autophagosome, and then they fuse with lysosomes for degradation and recycle of the engulfed components within the autolysosome to maintain cellular homeostasis. In male testis, the Leydig cells provide the major source of testosterone production. Autophagy is extremely active in Leydig cells and is involved in the steroid production. However, the precise role of autophagy in Leydig cells is still largely unknown. Thus, a comprehensive measurement of autophagic activity with different methods would shed light on our knowledge about the functional role of autophagy in regulating male reproductive physiology. In this chapter, we describe the morphological, cellular, and biochemical methods to monitor autophagy in Leydig cells.