Reduction or elimination of traditional hazardous chemicals in chromatographic separation and sample preparation is comparatively safer and can be considered as a greener approach for the determination of analytes in biological matrices. A rapid and sensitive UHPLC-MS/MS assay for determination of quetiapine in rat plasma was developed and validated using environment friendly and low toxic organic solvents as organic modifier in mobile phase and sample preparation. Quetiapine and risperidone (internal standard) were separated on Acquity BEH™ C18 column (50×2.1mm, i.d. 1.7μm) using ethanol–water-formic acid (80:20:0.1, v/v/v) as mobile phase at a flow rate of 0.3mL/min. Detection was performed on tandem mass spectrometer using positive electrospray ionization source by multiple reaction monitoring mode. The precursor to product ion transitions of m/z 384.13>253.00 for quetiapine and m/z 411.18>191.07 for IS was used to quantify them respectively. The method was found to be linear in the concentration range of 0.45–500ng/mL. All validation parameters were within acceptable range, as defined by guidelines of bio-analytical method validation. In conclusion, bioanalytical assay using UHPLC-MS/MS together with environment friendly and low toxic organic solvents in mobile phase and sample preparation may be considered as greener approach for quantification of analytes in plasma samples.