The aim of this study was to determine the thickness of the hydrodynamic diffusion layer (hHDL) of three poor water-soluble compounds under laminar fluid flow using a single particle dissolution technique. The single particle dissolution experiments were performed in a flowing aqueous medium using four different fluid velocities (v), ranging from 46 to 103 mm/s. The particles used had an initial radius (r) of 18.8 to 52.3 μm. The determined hHDL values were calculated from both dissolution experiments and computational fluid dynamics (CFD) simulation. In this study, single particle dissolution experiments gave, with one exception, hHDL values in the range of 2.09 to 8.85 µm and corresponding simulations gave hHDL values in the range of 2.53 to 4.38 µm. Hence, we found a semi-quantitative concordance between experimental and simulated determined hHDL values. Also, a theoretical relation between the dependence of hHDL on particle radius and flow velocity of the medium was established by a series of CFD simulations in a fluid velocity range of 10–100 mm/s and particle size (radius) range of 5–40 µm. The outcome suggests a power law relation of the form hHDL∝r3/5v-2/5. In addition, the hHDL seems to be independent of the solubility, while it has a diffusion coefficient dependence. In conclusion, the hHDL values were determined under well-defined conditions; hence, this approach can be used to estimate the hHDL under different conditions to increase the understanding of the mass transfer mechanisms during the dissolution process.