The efficient extraction of proteins from purified cellular organelles is critical for in vitro analyses, including identification of protein complex members by affinity purification-based quantitative proteomic approaches. When applied to purified nucleoli, classic nuclear protein extraction methods inefficiently and selectively release only approximately 50% of proteins. Here, we present a method that can extract up to 90% of nucleolar proteins, and apply it in a quantitative interactomic approach to identify nucleolar interaction partners for a mammalian protein phosphatase.