Teicoplanin (TEIC) and vancomycin hydrochloride (VAN) are glycopeptide antimicrobial drugs against methicillin-resistant Staphylococcus aureus and Gram-positive organisms. So, simple, quick, cost-effective and ultrasensitive spectrofluorimetric method was developed and bio-analytically validated for assay of TEIC and VAN. The proposed method is based on the reaction of acetylacetone with primary amine moiety in the presence of formaldehyde producing highly fluorogenic product measured at 485 nm (λex at 420 nm). The calibration range was constructed from (50–400 ng mL−1) and (70–800 ng mL−1) for TEIC and VAN, respectively. The presented method exhibited low LOD (7.04 and 3.55 ng mL−1) and LOQ (21.33 and 10.77 ng mL−1) values for TEIC and VAN, respectively. The presented method was effectively applied to determine TEIC and VAN in real human plasma with percentage of recovery ranged from 95.57 ± 2.06% to 97.43 ± 1.66% for the studied drugs. Also, the developed method was applied for the first time to study synthetic mixtures of the studied drugs with rifampicin (RIF), as a combination therapy to treat drug-resistant strains of Staphylococcus aureus without interferences from RIF. The synthetic mixtures of TEIC or VAN with RIF were prepared in three different ratios in order to measure the stability of the studied drugs TEIC or VAN in presence of RIF in pure forms and human plasma.