Trypanosoma cruzi is the etiological agent of Chagas’ disease, a neglected tropical disease affecting six to eight million people worldwide. The infection has a chronic progression, with cardiac, digestive, or neurological pathologies typically manifesting 10 or more years after the initial infection. However, during the first weeks of infection, the parasite colonizes nearly all tissues and organs of the mammalian host. The circulating trypomastigote stage is responsible for this systemic dissemination. After extravasation, trypomastigotes migrate through the interstitial matrix of target tissues to locate and invade nucleated cells. Although the mechanisms underlying this process are not yet fully elucidated, parasite migration and motility in a three-dimensional environment play crucial roles. This chapter discusses the technical aspects of monitoring trypomastigote motility using 3D collagen-I matrices and time-lapse microscopy. It begins with a straightforward method for isolating collagen-I (Col-I) from rat tail tendons. Next, a protocol is provided to assess the correct co-polymerization of Col-I and trypomastigotes into a three-dimensional structure using confocal reflection microscopy. Finally, we describe the monitoring and analysis of trypomastigote motility patterns through time-lapse microscopy. This protocol enables the real-time observation of live trypomastigotes, facilitating the study of their dynamics within a three-dimensional extracellular environment that closely mimics in vivo conditions. Additionally, the model allows the analysis of trypomastigote trajectories, movement types, and speed. Our protocol provides a valuable platform for investigating T. cruzi trypomastigote motility, offering insights into their dissemination strategies and potential interactions with host tissues.