The aim of this study was to assess the role of serum S100B protein as a biomarker for cardiovascular effects in an anesthetized rat model of amitriptyline toxicity. Adult male Wistar rats (n = 28) were randomized into four groups. While the control group received normal saline, the experimental groups received different doses of amitriptyline (0.625 or 0.94 or 1.25 mg/kg/min) infusion. Mean arterial pressure (MAP), heart rate (HR), electrocardiogram parameters, and serum S100B protein levels were recorded during the experiment. Linear Pearson's correlation coefficient was used to examine the association between cardiovascular parameters and serum levels of S100B protein. In the experimental groups, amitriptyline caused a significant decrease in MAP and HR (P < 0.001), a prolongation in QRS duration and QT intervals (P < 0.01), but it did not change PR intervals significantly. At the end of the experiment of the second group, a significant correlation was found between HR and serum S100B protein levels (r = -0.963, P = 0.037). At the end of the experiment of the third and fourth groups, a significant correlation between MAP, HR, all ECG parameters, and serum S100B protein levels was found. Serum S100B protein levels correlate well with amitriptyline-induced cardiovascular toxicity and can be used as a biomarker for predicting cardiovascular toxic effects of amitriptyline.