The invention discloses monoclonal antibodies for vitamins A (retinol palmitate) and D3 (cholecalciferol); a method for using monoclonal antibodies, and the monoclonal antibodies disclosed herein, in particular, to quantitate these vitamins in fluids such as dairy products, and blood, and also raw or processed agri-food and beverage products. The method involves contacting the sample with a mixture of polar and non-polar organic solvents in combination with inorganic salts to remove fat molecules into an organic fraction, and assaying the organic fraction by immunoassay involving the monoclonal antibodies. The presence of a mixture of non-polar and polar organic solvents increases the separation of vitamins from fat molecules and enables the test samples to be quantified by immunoassay without any further treatment.