Aim: To assess the blood glucose concentration in EDTA/F plasma and serum samples at a clinical laboratory. Materials and Methods: A cross-sectional observational study was conducted at our hospital to evaluate the glucose concentrations in blood samples collected under different conditions. A total of 50 participants were included in the study, regardless of their age, gender, fasting status, or underlying medical conditions. Each participant's blood glucose levels were measured on four different occasions, with samples collected simultaneously but processed under varying conditions.All blood samples were collected in the laboratory's phlebotomy room, ensuring immediate processing and eliminating any delays in transportation or exposure to environmental temperature fluctuations. Glucose concentrations were determined spectrophotometrically using the Randox Imola auto-analyzer (Randox Laboratories Limited, UK) via the glucose oxidase-peroxidase method. Results: Initially, at 30 minutes post-collection, the mean glucose concentration in plasma (P1a) was 102.5 mg/dL, slightly higher than the serum (S1a) concentration of 100.2 mg/dL. The standard deviations were similar, indicating comparable variability in measurements for both sample types. The plasma mean dropped to 101.0 mg/dL, and the serum mean to 98.7 mg/dL. These changes indicate that glucose concentrations can slightly decrease over time, even when samples are stored properly at 4°C, though the variability remained similar.For samples that were subjected to delayed centrifugation (P2a and S2a), the mean glucose levels were slightly lower than those processed immediately, with plasma at 100.0 mg/dL and serum at 97.5 mg/dL. The consistent pattern of plasma having slightly higher glucose concentrations than serum persisted across all time points. Conclusion: For blood glucose measurement in serum to be considered valid, the separation of serum by centrifugation must be completed within a maximum of 30 minutes. Using serum tubes has the benefits of avoiding excessive blood draws and improving turnaround time. Every collection facility must be equipped with a centrifuge to ensure timely blood separation.