Various ³¹P NMR techniques, including ³¹P-T₁, ³¹P-T₂, ³¹P-diffusion ordered spectroscopy and ³¹P{¹H}saturation transfer difference techniques, were applied to analyze the affinity of adenosine 3’-monophosphate (3’-AMP) to human serum albumin. To date, no structures of the complex comprising 3’-AMP and human serum albumin have been deposited in Protein Data Bank. Because the ³¹P nucleus is a selective marker of phosphorylated compounds, its observation by NMR spectroscopy can be an effective method to investigate these molecular interactions in the solution state. In the present system, the ¹H/³¹P-T₂ measurements provided the sensitive results that confirmed complex formation, and simple comparisons of the ³¹P signal decay using the pulse sequence to measure ³¹P-T₂ reflected these molecular interactions. The ³¹P{¹H} saturation transfer difference experiment was also applied to confirm 3’-AMP bound to proteins.