Abstract Gut microbiota play an important role in extraction, synthesis and absorption of nutrients. Commensal bacteria prevent pathogenic bacteria colonization and maintain intestinal epithelium integrity. The most common families of commensal bacteria in nursery pigs are Prevotellaceae, Clostridiaceae, Erysipelotrichaceae, Lachnospitaceae, Lactobacillaceae, Ruminicoccaceae and Streptoccocaceae. Understanding the microbial abundance shifts that causes health disruption leading to diarrhea and stunted growth performance can be of great benefit for developing mitigation strategies. Next generation sequencing (NGS) technology facilitates metagenomic approaches, developing sequencing profile representing any and all organisms within a sample. Electronic-probe Diagnostic Nucleic acid Analysis (EDNA) is a bioinformatic tool originally developed to detect species-specific plant pathogen targets in metagenomic databases. EDNA has been shown to reduce time to detect microbial signatures in large metagenomic sequence data. However, it has not previously been used as a metagenomic tool for assessing microbiome composition at the family level. Therefore, a metagenomic sequencing based in silico detection of gut microbiota using E-probes of the seven most common commensal families was developed and further validated in vitro. E-probes were designed from the selected families as follows, Prevotellaceae (89,565), Clostridiaceae (58,554), Erysipelotrichaceae (195), Lachnospitaceae (87), Lactobacillaceae (211,507), Ruminicoccaceae (14,575) and Streptoccocaceae (54,632). Fecal metagenomes of nursery pigs from 0, 7, 14, and 21 d were used to validate the E-probes. The hits were able to detect the relative abundance variations of the 4-time periods. The results between hits and reads were as follows, Prevotellaceae (r2 = 0.98), Clostridiaceae (r2 = 0.99), Erysipelotrichaceae (r2 = 0.99), Lachnospitaceae (r2 = 0.99), Lactobacillaceae (r2 = 0.91), Ruminicoccaceae (r2 = 0.99) and Streptoccocaceae (r2 = 0.98). These results validate in silico usage of E-probes to detect the relative abundance variations in gut microbiota. Further in vitro validation will be performed to assess the microbial changes related to diet in nursery pigs.