Evaluating the composition of the human gut microbiota greatly facilitates studies on its role in human pathophysiology, and is heavily reliant on culture-independent molecular methods. A microarray designated the Human Gut Chip (HuGChip) was developed to analyze and compare human gut microbiota samples. The PhylArray software was used to design specific and sensitive probes. The DNA chip was composed of 4,441 probes (2,442 specific and 1,919 explorative probes) targeting 66 bacterial families. A mock community composed of 16S rRNA gene sequences from intestinal species was used to define the threshold criteria to be used to analyze complex samples. This was then experimentally verified with three human faecal samples and results were compared (i) with pyrosequencing of the V4 hypervariable region of the 16S rRNA gene, (ii) metagenomic data, and (iii) qPCR analysis of three phyla. When compared at both the phylum and the family level, high Pearson's correlation coefficients were obtained between data from all methods. The HuGChip development and validation showed that it is not only able to assess the known human gut microbiota but could also detect unknown species with the explorative probes to reveal the large number of bacterial sequences not yet described in the human gut microbiota, overcoming the main inconvenience encountered when developing microarrays.