This book constitutes the refereed proceedings of the Second International Workshop on Medical Optical Imaging and Virtual Microscopy Image Analysis, MOVI 2024, held in conjunction with the 26th International Conference on Medical Imaging and Computer-Assisted Intervention, MICCAI 2024, in Marrakesh, Morocco, in October 2024. The 21 regular papers presented at MOVI 2024 were carefully reviewed and selected from 29 submissions. They are grouped into these two topical sections: Medical Optical Imaging and Virtual Microscopy Image Analysis and Kidney Pathology Image segmentation (KPIs) Challenge.