Stress is identified as a cause of transient hyperprolactinemia, whereas venipuncture is considered a source of stress for patient. The aim of this study was to investigate the association of venipuncture-induced stress with elevation of serum prolactin. This was a cross-sectional observational study conducted on a group of 150 outdoor patients visiting a tertiary care hospital. Serial sampling was performed by drawing venous blood at different time intervals (0, 30 and 60 min) by single venipuncture to measure serum prolactin to diagnose stress-induced hyperprolactinemia. The study was conducted in two phases, namely, Phase 1 and Phase 2, at different times. The Phase 1 results were divided into two groups: Group 1 (0 min) and Group 5 (pool prepared from samples collected at 0 + 30 + 60 min). Likewise, the results of Phase 2 were segregated into five groups; Group 1 (0 min), Group 2 (30 min), Group 3 (60 min), Group 4 (average of three groups), and Group 5 (pool from samples collected at 0 + 30 + 60 min). In both Phase 1 and Phase 2 of the study, there was a statistically significant (p = 0.0003 in Phase 1 and p = 0.02 in Phase 2) decrease in the mean prolactin (17.99 ± 24.76 ng/mL in Phase 1 and 19.61 ± 23.42 ng/mL in Phase 2) in the pooled samples (Group 5) in comparison to the mean prolactin (19.67 ± 27.69 ng/mL in Phase 1 and 21.06 ± 25.06 ng/mL in Phase 2) of the serum collected at 0 h (Group 1). There was no significant difference in the mean prolactin measured from the pooled samples and average prolactin calculated after individual testing from each sample collected at 0 h, 30 min and 60 min. Venipuncture-triggered fear and apprehension may result in transient hyperprolactinemia. In comparison to performing multiple testing on the samples collected at different time intervals and determining the mean, measurement of the analyte from the pooled serum is the better alternative as it can conserve both time and resources.