Deep sequencing techniques used in metagenomic approaches have greatly advanced the study of microbial communities in various environments. However, one microbial segment that has remained largely unexplored is the natural plasmids residing within microbial environments. Plasmids are perceived as mobile genetic elements that exist extra-chromosomally and occasionally carry accessory genes that confer an advantage to their host in its ecological niche. They are thus thought to play an important evolutionary role in microbial communities by laterally introducing genes and traits into microbial genomes. Despite their importance, technical obstacles still limit the metagenomic study of natural plasmids using deep sequencing techniques. These include low copy number of the plasmids and heterogeneity of microbes in environmental samples, reflected in the low abundance of each individual plasmid. Furthermore, the extracted plasmids usually contain remnants of chromosomal DNA that can potentially interfere with the analysis of unique plasmid traits. We have recently studied the rumen metagenomic plasmid population using a newly developed procedure that successfully overcomes these obstacles. This procedure enables extraction of pure plasmid DNA suited for deep sequencing studies. Here we present a detailed description and characterization of this procedure which could potentially allow the study of plasmids in other environmental niches.