We generated a detailed time series of total dissolved hydrolyzable amino acids (DHAA) in a watershed dominated by irrigated agriculture in northern California, USA to investigate the roles of hydrologic and seasonal changes on the composition of dissolved organic matter (DOM). DHAA are sensitive indicators of the degradation state and reactivity of DOM. DHAA concentrations ranged from 0.55 to 9.96 μM (median 3.51 ± 1.80 μM), with expected peaks during high-discharge storms and unexpected high values throughout the low-discharge irrigation season. Overall, summer irrigation was a critical hydrologic regime for DOM cycling since it mobilized DOM similar in concentration and reactivity to DOM released during storms. Together, irrigation and storm flows exported DOM with (1) the largest DHAA contributions to the dissolved organic carbon and the dissolved organic nitrogen pools, (2) the largest proportion of basic amino acids, and (3) the lowest degradation extent based on multiple indices. In this highly disturbed terrestrial system, UV–vis absorbance did not correlate with DHAA concentrations, while classic interpretations of common amino acid indicators (e.g., proportion of basic amino acids, degradation index, percent of non-protein amino acids) were prone to conflicting characterizations of DOM reactivity. Therefore, a new parameter (processing ratio, PR) derived from individual amino acid concentrations was developed that demonstrated a strong potential for mechanistic-driven characterization of the extent of DOM diagenesis in freshwaters. Irrigated agriculture altered stream biogeochemistry by releasing a continuous supply of reactive DOM (lowest PR values), thereby providing an additional energy source to downstream ecosystems.