¹Department of Pharmaceutical Chemistry and Pharmacognosy, Faculty of Pharmacy, Al-Azhar University-Gaza, Gaza, Palestine DOI : 10.29228/jrp.893 Synthetic colorants (SCs) are a class of pharmaceutical excipients, which could have harmful side effects especially in a vulnerable population like children. This study aimed to develop and validate a simple reverse phase high performance liquid chromatography (RP-HPLC) to quantitate eight SCs e.g. Tartrazine, Indigo carmine, Sunset yellow, Allura red AC, Carmosine, Ponceu 4R, Fast green FCF, and Brilliant blue FCF in pediatric over the counter medications (OTCs) and to assess the daily colorants intake through OTCs administration. After a solid phase extraction (SPE) using ChromabondHR-XAW cartridges, a gradient chromatographic elution was carried out using C18 column, and diode-array detector. Mobile phase composed of 80 mM ammonium acetate (pH 7.0) and methanol:acetonitrile 70:30 (v/v) solutions. Validation was performed according to ICH guidelines. The developed method was applied to quantify SCs in pediatric OTCs (syrups, suspensions, gummies, and chewable tablets) marketed in the Gaza Strip. Separation was completed within 18 minutes. The recovery rates of colorants were in the range of 72.01 - 117.15%. The developed method was linear with a correlation coefficient (R2 ˃ 0.9989). Limits of detection (LOD) and limits of quantitation (LOQ) ranged between 0.007 - 0.12, and 0.02 - 0.36 (μg/mL), respectively. The method showed unique selectivity, accuracy, and precision. Application of the developed HPLC method to assay synthetic colorants in different OTCs was successful. Assessment of daily colorant intake showed that the patients were exposed to 45% of the acceptable daily intake (ADI) of sunset yellow through an OTC. The ratio of SCs should be reduced to a minimum in pediatric formulations. Keywords : Synthetic colorants; HPLC-DAD; validation; OTCs; ADI