Live-cell assays to measure cellular function performed within 3D cultures have the potential to elucidate the underlying processes behind disease progression and tissue formation. Cells cultured in 3D interact and remodel their microenvironment and can develop into complex structures. We have developed a transcription factor (TF) activity array that uses bioluminescence imaging (BLI) of lentiviral delivered luminescent reporter constructs that allows for the non-invasive imaging of TF activity in both 2D and 3D culture. Imaging can be applied repeatedly throughout culture to capture dynamic TF activity, though appropriate normalization is necessary. We investigated in-well normalization using Gaussia or Renilla luciferase, and external well normalization using firefly luciferase. Gaussia and Renilla luciferase were each unable to provide consistent normalization for long-term measurement of TF activity. However, external well normalization provided low variability and accounted for changes in cellular dynamics. Using external normalization, dynamic TF activities were quantified for five TFs. The array captured expected changes in TF activity to stimuli, however the array also provided dynamic profiles within 2D and 3D that have not been previously characterized. The development of the technology to dynamically track TF activity within cells cultured in both 2D and 3D can provide greater understanding of complex cellular processes. Biotechnol. Bioeng. 2013; 110: 563–572. © 2012 Wiley Periodicals, Inc.