Invadopodia are specialized structures of cancer cells which aid in cancer cell invasion and metastasis. Therefore, studying the early steps of invadopodium assembly and its life cycle at the subcellular level by using high spatiotemporal resolution imaging provides an opportunity for understanding the signaling mechanisms involved in this very important process. In this chapter, we describe the design of a custom-built high-resolution fluorescence microscope which makes this challenging imaging possible. We also describe an ImageJ plugin that we have developed for tracking of invadopodia and lifetime analysis.