In this study, we introduce FrozONE, a workflow for the generation of deep nuclear proteomes by quick, robust and scalable nucleus enrichment from snap-frozen tissue material. We benchmarked our workflow’s capability to enrich and quantify nuclear proteins from multiple tissues and show its competitiveness with conventional ultracentrifugation-based nucleus enrichment strategies, while requiring shorter processing time, cheaper instrumentation and bypassing the need of fresh tissue processing, thereby simplifying experimental logistics and increasing scalability.