The combination of Raman spectroscopy and optical trapping holds great promise for single-cell studies and is an emergent theme in microfluidic environments. Here, the evolution of the Raman signal intensity with an axial increment of the mass of the substance of interest inside a specific Raman excitation volume is investigated. Whilst Raman spectroscopy may be applied to tissue samples, solutions and single cells, there are no easily available methods to rapidly acquire signals from small cell populations. We show a simple but powerful method to record the Raman intensity signal simultaneously from a small number of trapped cells or colloidal particles using the technique of optical stacking. The Raman spectra of stacks of red blood cells and yeast cells show that this method can be applied to biological systems. We demonstrate how we may reveal biochemical fingerprints that would otherwise require long integration times for each single cell or averaging over many sequentially acquired cell spectra. There is potential to apply this method to directly attain Raman spectra from sorted sub-populations of normal, abnormal and tumour cell lines. Copyright (C) 2007 John Wiley & Sons, Ltd.