Plants are valuable systems for analyzing the acentriolar mitotic spindle. We have developed methods for imaging the mitotic spindle in living tobacco (Nicotiana tabacum) suspension culture cells expressing GFP-?-tubulin. The methods allow the spindle to be observed in living cells at high spatial and temporal resolution and rely on water immersion objectives, spinning disk optics, and high-sensitivity cameras. Here, we describe these methods and provide step-by-step protocols for certain key steps. We also describe a method for application and removal of inhibitors.