Background Detecting total cholesterol in dried blood spots could aid in identifying individuals with a high likelihood of familial hypercholesterolemia and could be used as a screening measure. This study aims to assess the diagnostic accuracy of dried blood spots on Whatman 903 paper cards using a manual enzymatic technique. Methods: A total of 394 samples were collected as serum and dried blood spots were compared. Cholesterol was determined in serum using the automated reference method, while cholesterol on paper was measured using a manual enzymatic method. Within- and between-day diagnostic variability were analyzed. The correlation between both methods was assessed using Passing–Bablok regression and Bland–Altman plot. Internal validation of our correlation formula was performed on 149 samples, along with external validation of the formula proposed by Corso et al. Results: The within- and between-day coefficient of variation was found to be lower than 10.14% and 14.09%, respectively. Passing–Bablok regression indicated a precision of 0.803 and an accuracy of 0.96. Internal validation precision was measured at 0.716. The resulting positive and negative predicted values were 0.77 and 0.92, respectively, vs. 0.46 and 0.96 from the external formula. Conclusions: Total cholesterol analysis in dried blood spots demonstrates high precision and reproducibility. This method reliably enables the incorporation of this biological marker into neonatal screening for familial hypercholesterolemia detection.