Objectives Individual sensitivity to many widely used drugs is significantly associated with genetic factors. The purpose of our work was to develop an instrument for simultaneous determination of the most clinically relevant pharmacogenetic markers to allow personalized treatment, mainly in patients with cardiovascular diseases. Methods Multiplex one-step polymerase chain reaction (PCR) followed by hybridization on a low-density biochip was applied to interrogate 15 polymorphisms in the following eight genes: VKORC1 –1639 G>A , CYP4F2 1297 G>A , GGCX 2374 C>G , CYP2C9 *2,*3 (430 C>T, 1075 A>C) , CYP2D6 *3,*4, *6, *9, *41 (2549delA, 1846 G>A, 1707delT, 2615_2617delAAG, 2988 G>A), CYP2C19 *2,*3,*17 (681 G>A, 636 G>A, −806 C>T) , ABCB1 (3435 C>T) , SLCO1B1 *5 . Results Two hundred nineteen patients with cardiovascular diseases (CVD) and 48 female patients with estrogen receptor (ER)-positive breast cancer (BC) were genotyped. Of the 219 CVD patients, 203 (92.7%) carried one or more actionable at-risk genotypes based on VKORC1/CYP2C9, CYP2C9, CYP2C19, SLCO1B1, and CYP2D6 genotypes. Among them, 67 patients (30.6%) carried one, 58 patients (26.5%) carried two, 51 patients (23.3%) carried three, 26 patients (11.9%) carried four, and one patient (0.4%) carried five risk actionable genotypes. In the ER-positive BC group 12 patients (25%) were CYP2D6 intermediate or poor metabolizers. Conclusions The developed biochip is applicable for rapid and robust genotyping of patients who were taking a wide spectrum of medications to optimize drugs and dosage and avoid adverse drug reactions in cardiology, oncology, psychiatry, rheumatology and gastroenterology.