Herpes simplex virus (HSV)-1 is a large, enveloped DNA virus that undergoes a complex assembly pathway in the cell. Capsids assemble in the nucleus, cross the nuclear envelope into the cytoplasm by budding and fusion through the nuclear membranes, and acquire a proteinaceous ‘tegument’ layer in the cytoplasm before becoming enveloped at trans-Golgi network vesicles and/or recycling endosomes. Although the virus assembly pathway has been studied in detail using fluorescence and electron microscopy, several aspects of the process remain poorly understood. The remodelling of cellular organelles during HSV-1 infection has not been robustly quantitated using three dimensional (3D) volumetric analysis and the involvement of different cytoplasmic vesicles in envelopment remains to be differentiated. Furthermore, the precise roles of multiple viral gene products that are important for HSV- 1 assembly remain to be fully characterised. I used cryo-soft-X-ray tomography to image the ultrastructure of cryopreserved infected cells in 3D. In order to analyse these tomograms I developed Contour, a program to semi-automate the segmentation and quantitation of features from cryo-soft-X- ray tomograms. I characterised the role of different viral proteins in assembly by infecting cells with recombinant HSV-1 lacking expression of specific proteins and monitoring virion assembly in cells at late stages of infection. Super-resolution structured illumination microscopy allowed me to distinguish between unenveloped and enveloped virus particles in these cryopreserved samples. Tomographic X- ray imaging of HSV-1-infected cells allowed me to capture and characterise different stages of HSV-1 assembly in 3D. Additionally, confocal microscopy was used to probe the differential roles of TGN46- and transferrin receptor (TfR)-positive vesicles in the cytoplasmic envelopment of HSV-1 capsids. Striking changes occur to the morphology of mitochondria and the organisation of cytoplasmic vesicles and lipid droplets during HSV-1 infection. Comparative tomographic analysis offer insight into the roles of pUL16, pUL21, pUL34, VP16, and pUS3 in HSV-1 nuclear egress, and the roles of pUL11, VP16, pUL51, gK, and gE in cytoplasmic envelopment. Furthermore, this analysis indicates that envelopment occurs by budding of tegument-associated capsids into glycoprotein-containing patches of cellular vesicles, as opposed to wrapping of thin lamellae around capsids. The confocal microscopy data analysis suggests that TGN46+ vesicles are essential for envelopment but TfR+ vesicles are only sometimes involved. This multi-modal 3D imaging strategy has enabled elaboration of outstanding questions regarding the assembly pathway of HSV-1.