In this study the affinity of three amino acids for the surface of non-stoichiometric hydroxyapatite nanoparticles (ns-nHA) was investigated under different reaction conditions. The amino acids investigated were chosen based on their differences in side chain polarity and potential impact on this surface affinity. While calcium pre-saturation of the calcium-deficient ns-nHA was not found to improve attachment of any of the amino acids studied, the polarity and fraction of ionized functional side groups was found to have a significant impact on this attachment. Overall, amino acid attachment to ns-nHA was not solely reliant on carboxyl groups. In fact, it seems that amine groups also notably interacted with the negative ns-nHA surface and increased the degree of surface binding achieved. As a result, glycine and lysine had greater attachment to ns-nHA than aspartic acid under the reaction conditions studied. Lastly, our results suggest that a layer of each amino acid forms at the surface of ns-nHA, with aspartic acid attachment the most stable and its surface coverage the least of the three amino acids studied.