Extracellular hydrolytic enzymes play a central role in marine carbon and nutrient cycling. Their activities are typically measured immediately after sample collection, limiting integration with omics analyses conducted on frozen samples. We assessed the effects of one month of storage at -20 °C and -80 °C on potential activities of leucine aminopeptidase (LAP), β-glucosidase (β-glu), N-acetyl-β-D-glucosaminidase (NAG), and alkaline phosphatase (AP) in estuarine and offshore samples from the Gulf of Maine. Activities of LAP, β-glu, and NAG were generally well preserved on frozen filters, particularly at -80 °C. In contrast, AP showed greater sensitivity to freezing, especially in offshore particle-associated samples. All enzymes exhibited greater activity losses in frozen whole water samples than on filters. These results demonstrate that frozen filters, particularly when stored at -80 °C, can preserve several ecologically relevant extracellular enzyme activities and provide a practical approach to link enzyme assays with omics measurements on the same samples.