The goal of this study was to determine the reference intervals (RIs) of calcium levels (Ca) in the blood of Libyan individuals. Blood samples were obtained from 120 healthy males and females through aseptic venipuncture. Informed consent was obtained prior to sample collection. Serum was extracted from the centrifuged samples and analyzed spectrophotometrically to determine calcium concentrations. The non-parametric percentile method was applied to establish the RIs of calcium, results showed the reference period is 7.8- 10.1 mg/dl. The study establishes RIs for serum calcium concentration between 7.8 mg/dl and 10.1 mg/dl in the Libyan population. These findings underscore the importance of developing geographically specific RIs, as RIs derived from manufacturer's kits or other populations may not accurately reflect the unique characteristics of the Libyan population. The RIs identified in this study are more relevant and appropriate for the Libyan context.