The use of Raman spectroscopy for the qualitative and quantitative analysis of dilute aqueous solutions is of interest to the biopharmaceutical manufacturing sector. However, the inherent weakness of the Raman effect, coupled with spectral variability due to spurious signals from sample holders, can produce significant problems for chemometric‐based high‐throughput assays. Therefore, there is a need for a multi‐well sample holder that ensures robust and repeatable measurements, in particular from dilute aqueous solutions such as cell culture media. Here we demonstrate the efficacy of a novel, electropolished, stainless steel multi‐well plate (SS‐MWP) sample holder with 96 wells for dilute aqueous solution analysis. A comprehensive study of the spectroscopic behaviour was carried out and comparisons made with multi‐well plates fabricated from polystyrene, polypropylene, and aluminium. A key factor in the validation studies is the use of intrinsically weak Raman scattering systems, e.g. water and dilute glucose solutions. The data collected show that the SS‐MWPs are much superior in terms of robustness, resistance to chemical attack, and measurement reproducibility and as such are the ideal sample holders for Raman analysis of dilute solutions. Copyright © 2010 John Wiley & Sons, Ltd.