We report an improved tandem mass spectrometric assay for retinoic acid (RA) applicable to in vitro and in vivo biological samples. This liquid chromatography tandem mass spectrometric (LC/MS/MS) assay for direct RA quantification is the most sensitive to date, with a 62.5 attomol lower limit of detection and a linear range spanning greater than 4 orders of magnitude (from 250 attomol to 10 pmol). This assay resolves all-trans-RA (atRA) from its endogenous geometric isomers, is applicable to samples of limited size (10–20 mg of tissue), and functions with complex biological matrixes. Coefficients of variation are as follows: instrumental, ≤2.6%; intraday, 5.2% ± 0.7%; interday, 6.7% ± 0.9%. In vitro capabilities are demonstrated by quantification of endogenous RA and RA production (from retinol) in primary cultured astrocytes. Quantification of endogenous atRA and its geometric isomers in 129SV mouse serum and tissues (liver, kidney, adipose, muscle, spleen, testis, and brain) reveals in vivo utility of the assay. The ability to discriminate spatial concentrations of RA in vivo is illustrated with C57BL/6 mouse brain loci (hippocampus, cortex, olfactory bulb, thalamus, cerebellum, and striatum), as well as with Lewis rat proximal/distal mammary gland regions during various morphological stages: virgin, early pregnancy (e7), late pregnancy (e20), lactating (day 4), involuting day 1, and involuting day 11. This assay provides the sensitivity necessary for direct, endogenous RA quantification necessary to elucidate RA function, e.g., in neurogenesis, morphogenesis, and the contribution of altered RA homeostasis to diseases, such as Alzheimer’s disease, type 2 diabetes, obesity, and cancer.