Liquid chromatography–triple quadrupole mass spectrometry (LC–MS/MS) is becoming an increasingly essential analytical technique in many fields, such as food, environmental, biochemical, pharmaceutical, and clinical chemistry. The testing matrices, such as plasma and serum, cannot usually be sent directly to the LC–MS/MS system because of the large number of unrelated substances in addition to target analytes in samples, such as cells, tissues, and proteins. Protein precipitation (PPT) is the most utilized pretreatment method in clinical analysis because of its ease of use; its performance always involves the separation of the liquid phase from the solid phase, in which the most commonly used manipulation is centrifugation. However, there is an upper limit of throughput for centrifugation, and the cost and difficulty of integrating centrifugation into automatic equipment are usually high. To solve the drawbacks of the current PPT method, we developed a cartridge structure that can omit the centrifugation step in PPT, which, in turn, can facilitate the incorporation of the PPT method into an automatic clinical pretreatment procedure for LC–MS/MS. We used vitamin D analysis as a representative application in our endeavor to develop a centrifugation‐free PPT method that can be easily applied to a new automatic clinical pretreatment procedure.