A method combining microscopy with image analysis for studying ink penetration of ink-jet prints has been proposed. Prints on paper substrates of various material compositions have been microtomed, imaged with optical microscopy, and analyzed with image processing. The penetration depth and distribution of the ink-jet dye in the paper, which depend on the material compositions of the substrates and printed ink volumes, are then obtained by means of statistical analysis. The study focuses on uncoated fine papers, including five types of hand-sheets and two commercial papers. It is observed that ink penetration can be significantly reduced by adding internal sizing (hydrophobizing) chemicals in the hand-sheet making, while it is relatively less affected by addition of filler and fluorescent whitening agent (FWA). Ink volume obviously influences the depth of its penetration, with depth increasing linearly for higher ink volumes but displaying a disproportionately shallow penetration for smaller applied volumes.