A methodology is described and applied for performing carbon mass balances across cellulase enzyme production processes using both soluble sugar and insoluble cellulose substrates. The fungus Trichoderma reesei was grown on either glucose, lactose, or cellulose in aerobic batch mode, and the evolution of the main carbonaceous components (cell mass, cellulose, soluble protein, adsorbed protein, sugars, and carbon dioxide) was followed. A variety of analytical techniques were utilized to measure these components, including (i) gravimetric analysis, (ii) near-infrared spectroscopy, (iii) bicinchoninic acid based soluble protein measurement, (iv) gas mass spectrometry and flow rate, (v) CHNS/O elemental analyses, and (vi) high-performance liquid chromatography. The combined set of measurements allowed carbon mass balances across the cellulase production process to be assessed to determine the consistency of the underlying kinetic data. Results demonstrate the capability to determine the levels and distribution of all major carbonaceous components during the cellulase production process on both soluble and insoluble substrates. Average carbon mass balance closures were near 100% during early stages (< 72 h) of the cultivations using glucose, lactose, or cellulose as the substrates, but carbon mass closures trended high later in the cultivation. Analysis of carbon allocation results suggests that an error in the gas mass flow rate measurement was the primary cause for carbon mass balance closures to exceed 110% late in the process.