Ultrasonic velocity, Density and Viscosity for ternary mixture of n-Butanol, Water and Acetic acid (C4H9OH+H2O+CH3COOH) (which is used as running solvent for TLC in peptide and peptidomimetic synthesis) have been measured at a constant temperature 289K at different concentrations by using Ultrasonic Technique. From experimentally obtained data other Acoustical parameters like Adiabatic Compressibility, Adiabatic Impedance, Intermolecular Free Length, Available Free Volume, Relaxation Time, Ultrasonic Attenuation, Wada’s constant, Rao’s constant, Vander waal’s constant, Internal Pressure, Gibb’s Free Energy and Enthalpy have been calculated. In this non-ideal mixture, non-linear variation of acoustical parameter with composition of the chemical system suggests the presence of weak molecular interaction.