Osteoclast lineage cells (OLCs), including osteoclast precursors (OCPs) and mature osteoclasts (MOCs), participate in bone remodeling and mediate pathologic bone loss. Thus, it is essential to obtain OLCs for exploring their molecular features in both physiological and pathological conditions in vivo. However, the conventional protocols for obtaining OLCs ex vivo are not only time-consuming, but also unable to capture the cellular status of OLCs in vivo. In addition, the current antibody-based isolation approaches, such as fluorescence-/ magnetic-activated cell sorting, are not able to obtain pure osteoclasts because no unique surface antigen for osteoclasts has been identified. Here, we develop a rapid protocol for directly isolating OLCs from mouse bone marrow through magnetic-activated cell sorting (MACS). This protocol can rapidly enrich OCPs and MOCs, respectively, depending on the expression of the distinctive surface markers at their differentiation stages. It is optimized to isolate OLCs from four mice concurrently, of which sorting procedure could be completed within ~5 h.Copyright © 2022 The Authors; exclusive licensee Bio-protocol LLC.