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The present work investigates bioactivity and biocompatibility of femtosecond (fs) laser surface-modified Ti6Al4V alloy (Ti-alloy). Self-aligned conical surface features were generated on Ti-alloy when laser irradiated employing a Ti:sapphire pulsed fs laser of wavelength 800 nm. Modification of surface chemical composition resulting from fs-laser irradiation of Ti-alloy was examined using Grazing incidence X-ray diffraction (GIXRD) technique and micro-Raman spectroscopy. Sub-oxide phase of titanium was detected on Ti-alloy surface post-fs-laser irradiation leading to increased oxygen vacancies on sample surface. For in vitro bioactivity tests, untreated and fs-laser-treated samples were immersed in simulated body fluid for 2 weeks. Evidence of hydroxyapatite deposition on both untreated Ti-alloy, as well as, fs-laser-treated Ti-alloy surfaces after in vitro tests were provided by scanning electron …