<p>In many fermentation studies, the production of hydrolytic enzymes from agricultural residues is traditionally maintained for about three months. This period has become standard practice, although it is supported more by convention than by experimental validation. In this investigation, twenty-four types of agro-waste were subjected to anaerobic fermentation, both individually and in mixed combinations, over a three-month span. The activities of six major enzymes namely: cellulase, amylase, xylanase, invertase, pectinase, and protease were determined at monthly intervals. To understand the biochemical progression of the process, the data were evaluated using principal component analysis (PCA), multivariate analysis of variance (MANOVA), and partial least squares (PLS) regression. These methods helped identify relationships between enzyme activity profiles and total reducing sugar (TRS) concentrations. The analyses revealed that enzyme production generally peaked within the first or second month of fermentation before declining, a trend consistent with substrate exhaustion and metabolic stabilization. PCA accounted for 56.6 percent of the total variability (PC1 = 37.6%; PC2 = 19.0%), while PLS regression showed a high predictive accuracy (R² = 0.89) using cross-validation. These findings indicate that the fermentation period can be considerably reduced without loss of enzyme yield. Overall, the study establishes a data-driven basis for determining optimal enzyme harvesting time from organic waste fermentations, thereby improving process efficiency and supporting more sustainable bioprocess operations.</p> Graphical Abstract <p></p>