The oral absorption and systemic translocation of particulate matter via the gastrointestinal tract has been shown by a number of laboratories using a wide variety of particles in different animal species. While there is debate on the magnitude of particle intestinal translocation, which is encumbered by the differing experimental protocols, particularly the method of quantitation of absorbed material, few have sought to examine the pharmacokinetic aspects of particle absorption. We describe in this communication the development of a simple and a rapid fluorometric assay of quantifying tissue-laden fluorescent nanoparticles that is able to isolate, detect and quantify the presence of two or more particle populations differing both in their size and fluorescent label. Six types of polystyrene nanoparticles incorporating different fluorescent markers were spiked in whole livers. The fluorophores were extracted using our previously developed method of freeze-drying the tissue and using chloroform as the extractive solvent. Only two types of particle populations, orange-labelled 40 nm and Fluoroscein-emitting 500 nm nanoparticles, were sufficiently recoverable and provided a high signal-to-noise ratio for further work. The amount of tissue and type of biological tissue type also impacted on the nanoparticle recovery and detection, reflecting, perhaps, the quenching effects of interacting tissue-derived molecules. In addition, the results also indicate that the use of nanoparticles incorporating fluorescent dyes that have emission over 500 nm overcome the tissue interfering autofluorescence for low doses of nanoparticles. The use of this fluorometric method has several advantages compared with other modes of quantitation in that it is rapid, non-radioactive and the marker is non-leaching. More importantly, it allows the simultaneous detection of multiple fluorophores such that two or more different fluorescent particle populations can be detected in the same sample. This may enable the uncharted area of pharmacokinetic parameters, such as the impedance, augmentation or site of gut uptake of differently sized particles to be studied.