Measurement of substrate pH entails procurement of the substrate solution and measurement of the solution pH. Acid-base reactions are completed at the time of testing. Determination of substrate pH during development of a titration curve is more complex because it involves initially the reaction of a base with the substrate. Five factors that can influence the resulting pH values were investigated in this study and include amount of water added to substrate, method to procure substrate solution for pH determination, chemical form of base used, time allowed for acid-base reaction and the addition of CaSO4. Substrate in this study consisted of 3 sphagnum peatmoss: 1 perlite (by volume) amended with wetting agent. Dolomitic limestone (6 g·L-1 substrate) was added to substrate for the water amount and solution procurement method experiments. Except for the water amount experiment, deionized water was added by weight to achieve 95% container capacity. Dishes were incubated at 20 °C for specified times. To identify the minimal level of water necessary to ensure complete contact between base and substrate for neutralization, additions equivalent to 95%, 100%, 120%, and 150% container capacity were tested. The 95% level proved adequate. The saturated media extraction and pour-through bulk solution displacement methods for pH determination resulted in higher pH measurements in the incubated substrate than the squeeze bulk solution displacement method. This indicated that the former two methods diluted the soil solution. The squeeze method was deemed most effective. NaOH resulted in higher pH endpoints than Ca(OH)2. This was apparently due to a higher affinity of Ca2+ for peatmoss exchange sites. Since Ca2+ is the predominant cation associated with liming materials for soilless substrates, Ca(OH)2 is more appropriate for titration. From the tested incubation times of 0, 2, 4, 8, 24, 48, and 96 hours, the duration of 24 hours was found to be adequate to allow complete reaction of base with substrate acidity. The best procedure for determining pH in a substrate titration situation included a water level of 95% container capacity, Ca(OH)2 base, an incubation time of 24 hours and the squeeze solution displacement method. The additional CaSO4 was not necessary. Chemical names used: calcium sulfate (CaSO4), sodium hydroxide (NaOH), calcium hydroxide [Ca(OH)2], calcium ion (Ca2+).