We propose a new method of through-focus scanning optical microscopy (TSOM) without a reference database, i.e., a model-less TSOM method. Building a TSOM reference database is time-consuming or even impractical in some TSOM applications that involve complex structures, such as 3D NAND, or irregular shapes such as defects. The proposed model-less TSOM method was used to determine just the height of defect particles, for the first time as far as we are aware. Defect height is the only relevant dimension for the display panel application. Specifically, we analyzed 40 organic light-emitting diode (OLED) surface defects using a lab-developed motion-free TSOM tool consisting of a 50× objective lens (numerical aperture (NA) 0.55), a 532-nm light source, an imaging detector with a 7.5-µm pitch, and a deformable mirror. The tool is in-line and capable of achieving high throughput non-destructively, both relevant features for industrial applications. We investigated linear regression relations between newly defined TSOM parameters (TSOM height, TSOM area and TSOM volume) and the defect heights, which were first measured by atomic force microscopy (AFM). Following defect classification based on in-focus images, we successfully found that the AFM height has a linear correlation with 50% TSOM height (H50%) within ± 20.3 nm (1σ) error over the range of 140 to 950 nm. The one-sigma error, i.e., 20.3 nm, was approximately λ/26 or 1/43 of the depth of focus (DOF) of the applied microscope.