Influenza A virus (IAV) replication within host cells relies on tightly controlled, dynamic interactions between viral and cellular components. Yet we have a limited understanding of the viral replication cycle at molecular resolution. Electron microscopy of resin-embedded samples has traditionally been employed to investigate viral replication in cells. However, this technique relies on chemical fixation of samples, dehydration, and resin embedding, which leads to loss of molecular details. Cryo-electron tomography emerged as an indispensable tool enabling the exploration of viral replication within vitrified samples, providing an unperturbed environment. Here, we provide a sample preparation protocol for in vitro and cellular cryo-electron tomography of isolated IAV, as well as for cryo-focused ion beam milling of IAV-infected cells at different stages of infection. The emphasis of this chapter is on infection conditions, grid handling, and adaptation of microscopy settings to the individual sample while adhering to biosafety regulations.