Liquid hot water pretreatment is considered to be a promising method for increasing biomass digestibility due to the moderate operational conditions without chemical additions. A necessary step towards the scalability of this pretreatment process is performing pilot plant trials. Upscaling was evaluated with a scaling factor of 500, by using 50 mL in the laboratory and 25 L in a pilot plant batch reactor. Pretreatment times were varied from 30 to 240 min, and temperatures used were 180–188 °C, while applying similar heating profiles at both scales. The initial mass fraction of poplar wood chips ranged from 10% to 16%. Liquid hot water pretreatment at laboratory and pilot scale led to analogous results. The acetic acid analysis of the liquid and solid fractions obtained after pretreatment indicated that complete deacetylation of poplar biomass can be achieved.