Labeling cells with superparamagnetic iron oxide (SPIO) nanoparticles provides the ability to track cells by magnetic resonance imaging. Quantifying intracellular iron concentration in SPIO labeled cells would allow for the comparison of agents and techniques used to magnetically label cells. Here we describe a rapid spectrophotometric technique (ST) to quantify iron content of SPIO-labeled cells, circumventing the previous requirement of an overnight acid digestion. Following lysis with 10% sodium dodecyl sulfate (SDS) of magnetically labeled cells, quantification of SPIO doped or labeled cells was performed using commonly available spectrophotometric instrument(s) by comparing absorptions at 370 and 750 nm with correction for turbidity of cellular products to determine the iron content of each sample. Standard curves demonstrated high linear correlation (R2 = 0.998) between absorbance spectra of iron oxide nanoparticles and concentration in known SPIO-doped cells. Comparisons of the ST with inductively coupled plasma–mass spectroscopy (ICP-MS) or nuclear magnetic resonance relaxometric (R2) determinations of intracellular iron contents in SPIO containing samples resulted in significant linear correlation between the techniques (R2 vs ST, R2 > 0.992, p < 0.0001; ST vs ICP-MS, R2 > 0.995, p < 0.0001) with the limit of detection of ST for iron = 0.66 µg ml−1 for 106 cells ml−1. We have developed a rapid straightforward protocol that does not require overnight acid digestion for quantifying iron oxide content in magnetically labeled cells using readily available analytic instrumentation that should greatly expedite advances in comparing SPIO agents and protocols for labeling cells. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.