Systems using immobilized enzymes are attractive for a wide range of industrial and medical applications because they allow for the fabrication of stable, reusable substrates with highly specific functionality. The performance of these systems is greatly dependent upon the orientation and conformation of the adsorbed enzymes. To investigate these relationships, we have developed and applied methods to quantitatively assess the secondary structure of adsorbed enzyme layers on planar surfaces using circular dichroism (CD) spectroscopy and evaluate their bioactivity using colorimetric assays. These combined measurements provide molecular-level insights regarding whether observed changes in adsorbed enzyme bioactivity are due to the adsorbed orientation of an enzyme or adsorption-induced changes in its conformation. Using this approach, we investigated the adsorption behavior of lysozyme (HEWL), xylanase (XYL), and glucose oxidase (GOx) on OH-, CH3-. NH2-, and COOH-terminated alkanethiol self-assembled monolayer (SAM) surfaces. The bioactivities of small enzymes HEWL and XYL had pronounced variations between the different SAM surfaces despite their structural stability, highlighting the role of adsorbed orientation on bioactivity. In contrast, GOx, which is a much larger enzyme, exhibited wide variations in both its structure and bioactivity after adsorption, with adsorption-induced conformational changes actually enhancing its bioactivity. These results provide new insights into protein-surface interactions at the molecular level and demonstrate that adsorption can either promote or inhibit bioactivity depending on how the surface chemistry influences the orientation and conformational state of the enzyme on the surface.