Alterations in the human gut microbiota caused, for example, by diet, functional foods, antibiotics, or occurring as a function of age are now known to be of relevance for host health. Therefore, there is a strong need for methods to detect such alterations in a rapid and comprehensive manner. In the present study, we developed and validated a high-throughput real-time quantitative PCR-based analysis platform, termed ‘GUt Low-Density Array’ (GULDA). The platform was designed for simultaneous analysis of the change in the abundance of 31 different microbial 16S rRNA gene targets in fecal samples obtained from individuals at various points in time. The target genes represent important phyla, genera, species, or other taxonomic groups within the five predominant bacterial phyla of the gut, Firmicutes, Bacteroidetes, Actinobacteria, Proteobacteria, and Verrucomicrobia and also Euryarchaeota. To demonstrate the applicability of GULDA, analysis of fecal samples obtained from six healthy infants at both 9 and 18 months of age was performed and showed a significant increase over time of the relative abundance of bacteria belonging to Clostridial cluster IV (Clostridia leptum group) and Bifidobacterium bifidum and concurrent decrease in the abundance of Clostridium butyricum and a tendency for decrease in Enterobacteriaceae over the 9-month period.