Astrocytes, integral components of the central nervous system, are increasingly recognized for their multifaceted roles beyond mere support cells. Despite their acknowledged importance, understanding the intricacies of astrocyte morphology and dynamics remains limited. Our study marks the first exploration of astrocytes using optical diffraction tomography (ODT), aiming to establish a solid foundation for their detailed characterization. It offers valuable insights into the morphological changes in postnatal rat cortical astrocytes over a 7-day in vitro period in a label-free manner. Through comprehensive analysis of 3D refractive index maps and shape characterization techniques, we elucidate the developmental trajectory and dynamic morphological transformations of astrocytes in culture. Specifically, our observations revealed increased area and transition to larger, flattened shapes, and alterations in cell volume and density, indicating shifts in cellular composition. Furthermore, by employing unsupervised clustering and pseudotime trajectory analysis, we tracked the morphological evolution of astrocytes from highly directional to evenly spread shapes. These results emphasize the dynamic nature of astrocytes. In addition, this analysis marks the first use of trajectory inference based solely on morphology for neural cell types. Future studies could employ ODT to examine the morphological dynamics and interactions of various neural cell types on other types of substrates.