The reliability of a prosthetic implant needs durability, biocompatibility, and osseointegration capability. Accomplishing these characteristics, Ti-6Al-4V alloy is the main used material for implant fabrication. Moreover, it can be processed by additive manufacturing technique, permitting to meet the needs of patience-tailored, often complex shaped, prosthesis topologies. Once an implant is realized, it is finished by machining operations and its osseointegration capability is heavily influenced by the resulting surface roughness. Consequently, the assessment of this latter is mandatory to evaluate the prosthesis durability. This paper presents the analysis of surface roughness of Ti-6Al-4V micro-milled specimens produced by plastic deformation, selective laser melting, and electron beam melting processes. A central composite design was employed for planning the cutting tests. The comparison between surface roughness results and its values for enhancing osseointegration, firstly permitted to individuate the range of micro-milling suitable applications, which have been individuated as ball joints, bone plates, and screws. Next, the statistical analysis of the experimental measurements allowed the identification of the most influential micro-milling parameters together with the determination of the mathematical models of surface roughness by response surface methodology. The good comparison among calculated and experimental results revealed the reliability of the model, allowing the prediction of achievable surface roughness once micro-machining parameters are selected, or their optimization as a function of a desired surface roughness value.