Liquid hot water pretreatment has been proposed as a possible means of improving rates of enzymatic hydrolysis of biomass while maintaining low levels of inhibitory compounds. Supplementation of liquid hot water pretreatment with dissolved carbon dioxide, yielding carbonic acid, has been shown to improve hydrolysis of some biomass substrates compared with the use of water alone. Previous studies on the application of carbonic acid to biomass pretreatment have noted a higher pH of hydrolyzates treated with carbonic acid as compared with the samples prepared with water alone. This study has applied recently developed analytical methods to quantify the concentration of organic acids in liquid hot water pretreated hydrolyzates, prepared with and without the addition of carbonic acid. It was observed that the addition of carbon dioxide to liquid hot water pretreatment significantly changed the accumulated concentrations of most measured compounds. However, the measured differences in product concentrations resulting from addition of carbonic acid did not account for the measured differences in hydrolyzate pH.