This research focuses on providing a detailed characteristic of non-metallic inclusions (NM's) in 316L stainless steels with and without Ca treatment after machining using different cutting speeds. The electrolytic extraction (EE) technique was used for three-dimensional determinations of the inclusion characteristics. Quantitative data from the fragile non-metallic inclusions (such as size, surface area, number) in chips obtained from different cutting speeds and materials were determined. The morphologies of NMIs in the chip samples were quite different compared to the original inclusions in the stainless steel samples before machining. It was proved that the deformation degree of soft inclusions such as MnS and CaO-SiO2-Al2O3-MgO-TiOx is dependent on the cutting speed as well as the temperatures and deformation degrees of the metal matrix during machining. The total surface areas of MnS inclusions increase from 2.8 to 3.8 times compared to the original total areas with an increased cutting speed. The total surface areas of soft oxide inclusions also increase from 1.1 to 3.5 times compared to the original total areas. In addition, the tool-chip contact lengths were also measured on the rake face of the tool, and the results were compared to the determined characteristics of the observed inclusions. It was found that the modification of NMIs by Ca treatment in 316L stainless steels is preferred for high cutting speeds.