As three-dimensional (3D) cell culture systems gain popularity in biomedical research, reliable assays for cell proliferation within 3D matrices become more important. Although many cell quantification techniques have been established for cells cultured on nondegradable plastic culture dishes and cells suspended in media, it is becoming increasingly clear that cell quantification after prolonged culture in 3D polymeric scaffolds imposes unique challenges because the added presence of polymeric materials may contribute to background signal via various mechanisms including autofluorescence, diffusion gradients, and sequestering effects