This instrument developmental research developed a pain scale for preterm neonates and to examine its psychometric properties. A developmental neurobiological approach was used to guide construction of the scale. Seven pain indicators were initially identified by the synthesis of data from a concept analysis, clinical observations, and expert interview; and were then reviewed for content validity by a panel of six experts. After the review, the respiratory support indicator was eliminated. Six indicators remained with two scoring formats for two age groups (< 32 weeks and ≥ 32 weeks to 36 weeks and 6 days) During psychometric testing, 53 blood collecting occasions from 19 preterm neonates in two neonatal intensice care units in Thailand were examined by two observers using the 6-indicator Clinical Pain Scale for Preterm Neonates scale and the Premature Infant Pain Profile-Revised scale. Prior to reliability analysis, length of the unit stay and previous pain exposure indicators were deleted because of low inter-item and item-total correlations. Cronbach’s alpha coefficient of the 4-indicator scale was 0.94 and the intraclass correlation coefficients ranged from 0.91 to 1.00. Construct validity was tested by comparing median pain scores of three phases. The results revealed that the median pain score of the puncture phase was significantly higher than those of baseline and recovery phases. Convergence examination showed a positive correlation between pain scores measured by the new scale and the Premature Infant Pain Profile-Revised Scale. Clinical utility evaluation of the new scale revealed satisfactory results. Thus, the Clinical Pain Scale for Preterm Neonates scale proved to be valid, reliable, and clinically applicable for procedural pain assessment in preterm neonates in neonatal intensive care unit.