Human bone marrow is a rich source of hematopoietic stem cells, mesenchymal stem cells (MSCs), and other progenitor cell types. These cells play a crucial role in regenerative medicine, cell-based therapies, and tissue engineering. The ability to efficiently isolate viable bone marrow mononuclear cells is essential for subsequent applications such as cell culture, transplantation, or molecular analysis. This protocol describes the step-by-step procedure for extracting human bone marrow from bone fragments, isolating mononuclear cells, and preparing the cell suspension for in vitro culture or further experimentation. The process includes initial washing to remove contaminants, maceration (if necessary) to break down tissue, and the use of density gradient centrifugation (Lymphoprep®) to separate the mononuclear cell fraction from other cell types such as red blood cells. The successful isolation of high-quality bone marrow cells is dependent on the careful execution of each step, including proper handling of reagents, centrifugation parameters, and maintaining sterile conditions to ensure cell viability and purity. This protocol provides a reliable method for isolating human bone marrow cells, which can be used for a variety of research and clinical applications, including stem cell research, immunotherapy, and disease modeling.