Current methods for assessing cell proliferation in 3D scaffolds rely on changes in metabolic activity or total DNA, however, direct quantification of cell number in 3D scaffolds remains a challenge. To address this issue, we developed an unbiased stereology approach that uses systematic-random sampling and thin focal-plane optical sectioning of the scaffolds followed by estimation of total cell number (StereoCount). This approach was validated against an indirect method for measuring the total DNA (DNA content); and theBürkercounting chamber, the current reference method for quantifying cell number. We assessed the total cell number for cell seeding density (cells per unit volume) across four values and compared the methods in terms of accuracy, ease-of-use and time demands. The accuracy ofStereoCountmarkedly outperformed theDNA contentfor cases with ~ 10,000 and ~ 125,000 cells/scaffold. For cases with ~ 250,000 and ~ 375,000 cells/scaffold bothStereoCountandDNA contentshowed lower accuracy than theBürkerbut did not differ from each other. In terms of ease-of-use, there was a strong advantage for theStereoCountdue to output in terms of absolute cell numbers along with the possibility for an overview of cell distribution and future use of automation for high throughput analysis. Taking together, theStereoCountmethod is an efficient approach for direct cell quantification in 3D collagen scaffolds. Its major benefit is that automatedStereoCountcould accelerate research using 3D scaffolds focused on drug discovery for a wide variety of human diseases.