The pour-through (PT) method is used in greenhouse and nursery production to monitor nutrient availability in soilless substrates. Efficacy of this method is based on the assumption that chemical properties of extracted solutions remain stable from the moment of collection until analysis. Extracted substrate solution can be analyzed directly in the greenhouse or sent to laboratories for complete nutritional analysis; thus, proper sample preservation methods (e.g., filtration and low temperatures) are critical for reducing sample contamination or degradation during storage. However, evidence of how these preservation methods affect chemical characteristics of PT samples is limited. The objective of this study was to evaluate the effect of storage time, storage temperature, and filtration of PT samples on pH, electrical conductivity (EC), and nutrient concentrations from pine bark– and peat-based substrates. PT extracts were obtained from liquid-fertilized fallow pots of either 100% milled pine bark (Expt. 1) or a 4 sphagnum peat: 1 perlite (by volume) substrate (Expt. 2). Aliquots of PT extract were either filtered or nonfiltered and then stored in plastic bottles at −22, 4, or 20 °C. EC, pH, and nutrient concentrations were analyzed at 0, 1, 7, and 30 days after PT sample collection. EC and pH in PT extracts of peat and pine bark, respectively, changed 1 day after collection. Storage time had the greatest effect on nutrient concentrations of samples stored at 20 °C. However, at day 30, nutrient concentrations had also changed in samples stored at 4 and −22 °C. Analytes that fluctuated most in both experiments and across all preservation treatments were dissolved organic carbon, total dissolved nitrogen, NO3−-N, and PO43−-P, whereas Ca2+, Mg2+, and SO42−-S were more stable in PT samples. This research suggests EC and pH should be analyzed immediately, whereas samples requiring nutrient analysis should be filtered immediately after collection, stored at 4 or −22 °C (preferably −22 °C), and analyzed within 7 days of collection.