This work proposes a rapid, simple and accurate analytical method for quantification of the organophosphate flame retardant triphenylphosphate (TPhP) and the biomarker diphenylphosphate (DPhP) in whole blood by a simple and effective sample preparation followed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) quantification. A liquid–liquid microextraction (µLLE) from 250 µL of mouse whole blood was developed and validated to quantify TPhP and DPhP. Nature of extraction solvent, grinding for cell lysis and purification step were investigated. The use of distilled solvents proved necessary to reduce and control the contamination of the ubiquitous target compounds throughout the analytical process and to maintain a stable and repeatable signal in the analytical controls. The validation was performed by matrix-matched calibration with a correction by labelled internal standards: the limit of quantification was 1 ng/mL for DPhP and 2 ng/mL for TPhP with good linearity over the range up to 48 ng/mL for each compound. Accuracy was between 92.5% and 106.7% with relative standard deviation < 20%. The µLLE-LC-MS/MS method was successfully tested during short exposures of mice to DPhP or TPhP via drinking water or by intravenous injection.