Microbial defense systems provide fundamental elements for biotechnology development. For example, molecular cloning techniques greatly depend on the discovery of the bacterial restriction-modification system, and similarly, deciphering the CRISPR adaptive immunity gives rise to the revolutionary genome editing technique CRISPR-Cas9. Therefore, exploring novel microbial defense systems has attracted world-wide attention, and new systems like prokaryotic Argonautes (pAgos) and the defense island system associated with restriction-modification (DISARM) were successively discovered and investigated. Recently, Sorek et al. reported a systematic strategy to predict novel defense systems within the vast microbial genomes and validated the antivirus or anti-plasmid capability of 10 candidates using synthetic biology procedures. Here, we introduce the systematic analysis pipeline developed by Sorek et al., summarize the characteristics of the novel microbial defense systems, and prospect the research trends and challenges in this rising field.