Changes in microbial biomass in the rhizosphere of young barley seedlings was studied. A fumigation-extraction (FE) method with measurement of ninhydrin-reactive nitrogen (NR-N) and a substrate-induced respiration (SIR) method were applied on a microscale to rhizosphere soil samples of approximately 0.1 g. Rhizosphere soil was defined as the soil adhering to the roots when they were carefully separated from the bulk soil. The rhizosphere soil was gently washed off the roots with either distilled water (FE) or with glucose solution (SIR). Shaking and mild sonication was used to disperse the soil without disrupting the roots. Fumigation was carried out by direct addition of liquid chloroform to the isolated soil. These techniques were proven to give reliable results under the experimental conditions of this investigation. Rhizosphere soil was isolated from segments of the roots representing different distances to the seed different root ages. In the rhizosphere of young barley seedlings, biomass NR-N increased significantly compared to the bulk soil from day 6 after sowing (average increases of 33–97%), especially where adventitious roots had developed. From this time, SIR rates were also significantly higher in the rhizosphere than in bulk soil (average increases 72–170%). The average ratio of SIR rate to biomass NR-N was found to be approximately 50% higher in the rhizosphere than in the bulk soil, which may indicate that a larger fraction of the microbial community is potentially active in the rhizosphere as compared to the bulk soil.