We acquired multiphoton images of normal and lung adenocarcinoma cell lines in three dimensions. Image stacks of the cells were then processed to obtain nucleus-to-cytoplasm (N/C) ratios in two and three dimensions. While N/C ratios in three dimensions can be unambiguously determined from the volumetric ratios of the nucleus and cytoplasm, two-dimensional (2-D) N/C can vary depending on the axial plane selected for N/C ratio determination. We determined 2-D N/C ratios from three criteria: (1) axial position at which the nuclear area is the largest; (2) the largest 2-D N/C ratio value; and (3) axial position at the midpoint of nuclear axial position. We found that different definitions of 2-D N/C ratio will significantly affect its value. Furthermore, in general, larger variance was found in 2-D rather than three-dimensional (3-D) N/C ratios. Lack of ambiguity in definition and reduced variance suggest that 3-D N/C ratio is a better parameter for characterizing tumor cells in the clinical setting.