A new, rapid, economical, and gradient reverse phase high-performance liquid chromatography (RP-HPLC) method has been developed and validated for the quantitative determination of Pimecrolimus in the bulk and pharmaceutical dosage form. The chromatographic separation was achieved with YMC Pack pro (C-18, 250×4.6 mm) and 5 µm particle size column. The optimized mobile phase consists of a phosphate buffer, pH adjusted to 2.5 with o-phosphoric acid and acetonitrile. The flow rate was 1 mL/min and eluents were detected at 210 nm using PDA detector. The retention time of Pimecrolimus was found to be 13 min. The percentage recoveries for molecules were found to be in the range of 99–101%. The calibration curve demonstrated good linearity in the range of 5–30 g/ml for Pimecrolimus. The degradation studies were conducted under a variety of stress conditions, including oxidation, thermal, photochemical, and ultraviolet radiation. The developed technique was approved in accordance with ICH recommendations. The validated method was suitable for the detection of Pimecrolimus in bulk drug substances and pharmaceutical dosage forms.