In-line graphene characterization to determine quality, area coverage fraction, and layer number on transparent substrates is critical to large-scale commercial graphene production. Many applications, including biosensors and imbedded diagnostics, flexible electronics, and transparent electrodes, require uniform graphene transfer from its native chemical vapor deposition foil to transparent films. To enable high-volume production of these devices, graphene layer number, quality, and area coverage must be mapped at high spatial resolution to enable growth and transfer process optimization. To this end, we present a spatially resolved optical transmission technique combined with statistical analysis of the measurements to determine graphene layer number on different transparent substrates, including polymer films and glass. This method can be automated and does not require user-inputted threshold values. Our method can effectively map >1 cm2 areas at 10 micron resolution and is not limited by type of substrate or thickness assuming the substrate is transparent. We corroborate these experimental results with simulated data and present guidelines to reasonably assess graphene quality, layer number, and feature size as functions of the experimental parameters.