This study investigates the application of a commercial low-field benchtop NMR for real-time monitoring of enzymatically catalyzed reactions, focusing on the diastereoselectivity of the threonine aldolase-catalyzed stereoselective aldol reaction between glycine and benzaldehyde. Despite the signal overlap inherent in the weak electromagnetic field of the benchtop NMR system, a complemental hard modeling (CHM) approach effectively differentiates between diastereomers, enabling the determination of enzymatic diastereoselectivity and the transition from kinetic to thermodynamic control. In particular, the achievement of thermodynamic equilibrium in the enzymatic aldol reaction is observed for the first time using in-line methods, occurring at 30% benzaldehyde conversion after 2 h. In-line NMR analysis reveals a diastereomeric excess of 37:63 (erythro/threo), which closely aligns with off-line measurements via GC and HPLC (36:64). This determination of diastereomers using CHM enhances the efficiency of in-line monitoring in enzymatic reactions, promising significant advancements in pharmaceutical process development. Overall, the study underscores the utility of benchtop NMR systems for in-line analysis of enzymatic reactions, offering insights into reaction mechanisms, selectivity, and equilibrium dynamics, thereby facilitating more efficient process optimization in the area of fine chemicals.