A simple and sensitive two-step reversed-phase HPLC method was developed and validated for determining amphotericin B, paracetamol and sulfapyridine in rat plasma using piroxicam as internal standard. This was subsequently employed in a pharmacokinetic study in rats following simultaneous oral administration of solid lipid nanoparticle formulations of amphotericin B, paracetamol and sulfasalazine. The paracetamol and sulfapyridine served as markers for estimating gastric emptying and cecal arrival times. A gradient elution was employed in the analysis and detection at 405 nm for amphotericin B and 254 nm for paracetamol and sulfapyridine. The internal standard was detectable at both wavelengths. The gastrointestinal study was conducted on male Sprague-Dawley rats. Calibration curves were linear for the three drugs (r 2 ≥ 0.9998) with the detection and quantification limits comparable with those in literature. Drug recovery from rat plasma was more than 92%. The accuracy of the method was < 8% for the three drugs whilst the precision was 10%. Paracetamol was rapidly absorbed from the small intestine reaching its C max in 1 hr while amphotericin B was absorbed slowly reaching peak levels in 8 hr. Sulfapyridine was absorbed only after a lag time of 2 hr due to delayed arrival and hydrolysis of sulfasalazine at the colon by azo-reductase bacteria. The HPLC method is rapid and applicable for the simultaneous estimation amphotericin B, paracetamol and sulfapyridine in rat plasma, which may serve for an indirect estimation of gastric emptying and orocecal transit properties of amphotericin B within dosage forms after oral administration.