Protein typically represents the largest input of organic nitrogen (N) into soil. Proteases subsequently make this protein available for use by both plants and microorganisms, however, the factors that regulate protein breakdown in the rhizosphere remain limited. Root exudation of carbon (C) and N into soil promotes microbial growth and thus enzyme production, which is further enhanced by root morphological traits such as root hairs. However, it is not clear how inputs of protein from external sources (e.g. necromass) affect enzyme activity in the rhizosphere. Insight into the interaction between protein addition and root morphology will enhance our knowledge of plant and microbial strategies for promoting N acquisition. Using soil zymography, we investigated the spatial distribution of leucine aminopeptidase activity in the rhizosphere of Hordeum vulgare L. (barley) with and without root hairs subject to localised protein addition. Seedlings of barley were grown for two weeks in rhizoboxes and soluble protein was applied 48 h before analysis of leucine aminopeptidase activity. In situ zymography was used to quantitatively visualise leucine aminopeptidase activity while ex situ sampling was used to determine its enzyme kinetics. In the zymograms, we found that mean and maximal leucine aminopeptidase activity was highest in the barley genotype with root hairs and in the presence of soil protein hotspots. This suggests that microorganisms and plant roots in the rhizosphere of genotypes with root hairs have a greater advantage in accessing protein hotspots in the soil. Leucine aminopeptidase activity did not follow the same trends when analysed by in situ zymography and ex situ sampling methods. Therefore, we recommend the use of in situ zymography to detect the spatial distribution of enzymatic hotspots and rhizosphere extent followed by ex situ sampling for assessing enzyme kinetics in the hotspot areas detected by in situ sampling. However, sampling biases must be considered to ensure enzyme activities are being interpreted as the true rhizosphere.