We have explored the use of X-ray microcomputed tomography (microCT) for assessing cell adhesion and proliferation in polymer scaffolds. Common methods for examining cells in scaffolds include fluorescence microscopy and soluble assays for cell components such as enzymes, protein or DNA. Fluorescence microscopy is generally qualitative and cannot visualize the scaffold interior. Soluble assays quantitatively measure cell number but do not yield information on cell spatial distribution. Herein, the ability of microCT to detect cells in scaffolds was compared with fluorescence microscopy and a soluble DNA assay. Comparisons were performed using polymer scaffolds that were seeded with cells at different densities and cultured for different times. The results showed that fluorescence microscopy had better resolution than muicroCT and that the soluble DNA assay was approximately 5x more sensitive than microCT under the conditions tested. However, microCT was able to image through opaque scaffolds to yield quantitative 3D imaging and analysis via a single, non-invasive modality. Quantitative microCT analysis of cell penetration into scaffolds was demonstrated. Further, quantitative microCT volume analysis required that the cell density in the scaffolds be greater than 1 million cells per mL indicating that microCT is best suited for quantifying cells at relatively high density during culture in scaffolds. In sum, the results demonstrate the benefits and limitations of using microCT for 3D imaging and analysis of cell adhesion and proliferation in polymer scaffolds.