A method and system for determining a biopolymer array substrate thickness dependent optimal focus distance for scanning a molecular array by a molecular array scanner are provided. A reference substrate is automatically scanned at successively greater distances of the stage from a light gathering medium, such as an optical fiber, or z-positions, to produce data providing a functional relationship between z-position and measured signal intensities. The data is then processed by an array substrate thickness dependent focus-finding routine that selects an optimal focus-distance for data scans which is optimized for the thickness of the reference substrate. Also provided are methods of determining the thickness of a biopolymer array substrate using a position sensitive device (PSD) component of a biopolymer array scanner. Further methods include determining the thickness of said biopolymeric array and automatically selecting an optimal focus distance using the determined thickness and a calibration function on thickness versus optimal focus distance. The subject invention finds use in a variety of different applications, including both genomic and proteomic applications.