The objective of this study was to establish normal reference ranges for porphyrins in healthy neonates. There is little information about urinary porphyrin excretion in this age group. This knowledge may provide an early diagnostic tool for detecting subtle alterations or latent forms in disorders of heme biosynthesis. Fifty healthy neonates were selected from the Department of Obstetrics. Total urinary porphyrins in random specimens were analysed by a spectrofluorometry method. The measurement of porphyrin fractions was made by fluorometric high-performance liquid chromatography (HPLC). The results were adjusted to urinary creatinine excretion to correct any imprecision and interindividual variation in body mass. The urinary total porphyrin had a median value of 331.50 (nmol/L). A statistically significant relationship between total porphyrin (nmol/L) and creatinine (mmol/L) was found (p < 0.01). The porphyrin/creatinine ratio showed a median value of 56.30 nmol/mmol creatinine. The study of individual porphyrins revealed that coproporphyrin and uroporphyrin were the major porphyrins excreted in neonates (coproporphyrin represents 81.98% and uroporphyrin 16.64% of total porphyrin); in both cases, isomer I was predominant with median values of 22.36 and 6.25 nmol/mmol creatinine, respectively. No significant relationships were found between porphyrin excretion and sex, gestational age, weight, or height. Our data provide the reference limits for porphyrins in neonates as a diagnostic guideline for evaluation of subtle alterations in heme biosynthesis.