Recent studies indicate that seed microbiomes affect germination and plant performance. However, the interplay between seed microbiota and plant health is still poorly understood. To get a complete picture of the system, a comprehensive analysis is required, comprising culture-dependent and culture-independent techniques. In this chapter, we provide a combination of methods that are established and optimized for the analysis of the seed microbiome. These include methods to: (1) activate and cultivate dormant seed microbiota, (2) analyze microbiota in germinated seeds (with and without substrate), (3) quantify microbial DNA via real-time PCR, (4) deplete host DNA for amplicon and metagenome analysis, and (5) visualize seed endophytes in microtomed sections using fluorescent in situ hybridization (FISH) and confocal laser scanning microscopy (CLSM). A deep understanding of the seed microbiome and its functions can help in developing new seed treatments and breeding strategies for sustainable agriculture.