Although many reports over the past few decades have documented protease activity within the nucleus of various eukaryotic cells and tissues, the identity and biological significance of these nuclear proteases remain largely unknown. In-gel zymography is an elegant, sensitive, and inexpensive technique to identify and investigate proteases within complex biological mixtures, including nuclear soluble extracts. We and others have successfully leveraged in-gel zymography with purified nuclear extracts to discover several novel nuclear proteases, including metalloproteinases and cysteine proteases. In this chapter, we provide a detailed protocol to purify nuclear extracts from cells and tissues, prepare and execute in-gel zymography, and determine the identity of the associated nuclear protease(s).