<p>Cover glasses are widely used in microscopy owing to their optical clarity and hydrophilic surfaces. However, their hydrophilicity can cause nonspecific biomolecule adsorption and liquid spreading. To address these challenges, hydrophobic modifications using poly(dimethyl siloxane) (PDMS) were induced via spin-coating at speeds from 500–4000 rpm. The PDMS layer thickness decreased with increasing spin speed, from 128.6 µm at 500 rpm to 13.32 µm at 4000 rpm. A hydrophobic surface was consistently achieved at speeds above 1000 rpm, with contact angles averaging 120°, whereas lower speeds resulted in uneven coatings. Although optical transparency decreased slightly with increasing thickness, it remained unaffected at higher spin speeds. Tribological analysis indicated that thinner coatings exhibited lower friction and improved durability, as evidenced by reduced average friction coefficients and narrower deviations at higher spin speeds. These findings can help optimize spin-coating parameters for fabricating hydrophobic cover glasses for advanced cell culture and imaging applications. Future studies will focus on integrating these coatings into practical cell observation systems.</p>