This chapter describes the isolation, expansion, staining, and imaging of osteoclasts from murine (MKS, BKD, and MMM) and human (NS, JBO, and KS) sources. We cover in detail both traditional and more modern methods of assessing osteoclast formation and function in vitro including advances in image acquisition and automated analyses. Importantly, we provide in-depth methods for human osteoclast culture systems, methods to assess human osteoclast function, and highlight potential methodological pitfalls and ways to overcome them. This collection of protocols provides a valuable resource for labs either initiating in vitro osteoclast assays or aiming to expand on traditional methods.