Matrigel is often used to study the growth of mammary epithelial cells with formation of either normal or cancerous characteristics that differs from the monolayer formation observed on 2D polystyrene. Morphological differences between 2D and 3D are associated with differential signaling, including differential response to E2 reported herein and decreased Rho and ERK activities resulting from decreased focal adhesions and tensional forces. AP1, CRE, and SRE had decreased activities over time and are downstream components of the MAPK pathway, which is influenced by Rho and ERK signaling. Hence, our results are consistent with decreased MAPK signaling for 3D relative to 2D culture. In addition to differences in growth, cells within 3D matrices may have an in vivo phenotypic response to chemotherapeutics, further supporting the value of 3D culture for analysis of cellular processes. Bioluminescence imaging was instrumental in enabling largescale, dynamic, quantitative measurement of TF activity. Results from bioluminescence imaging were consistent with the typical approach of cell extraction and lysis, a labor-intensive process that increased sample-to-sample variability. The normalization construct accounted for spot-to-spot variability in transfection, a variable that would otherwise hinder statistical analyses. Also, normalizing to the TA control construct was important to account for differences in transgene expression due to degradation and silencing of the reporter plasmids. Luciferase reporters provided a sensitive method due to enzymatic signal amplification, with signals detected over ICI 182780 several orders of magnitude. Dynamic analysis of TF activity in a single sample is more common with fluorescent protein reporters, which can be quantified by plate readers. The fluorescent reporters, however, lack signal amplification and thus have more limited detection of weak signals. Combining luciferase reporters with bioluminescence imaging provided both sensitivity and dynamic analysis, and captured the activity of numerous TFs simultaneously while minimizing the number of samples and reagents. Our 3D TF activity array profiled the activity of numerous signaling pathways simultaneously as cells organized into structures and responded to biochemical stimuli. Bioluminescence imaging was employed as a non-destructive technique capable of repeated measurement that enabled dynamic activity to be quantified. The array detected active TFs in at least 10 of the 28 pathways profiled, and the outputs of the array were consistent and reproducible. Importantly, this activity was captured dynamically, which identified pathways that were initially activated by a biochemical stimulus, and those pathways whose activity was altered subsequen.