Background: Antimicrobial resistance (AMR) is the ability of a pathogenic microbe to develop a resistance to the effects of an antimicrobial medication. AMR is one of the most concerning issues in medicine today, impacting morbidity, mortality and socioeconomic factors. Objective: To determine the prevalence and the phenotypic methods of detection of antibiotic sensitivity of gram-positive bacteria in clinical isolates collected from inpatients at the Sohag University Hospital during one year. Patients and methods: Fresh samples (blood, urine, sputum, pus and cerebrospinal fluid) were collected of 69 patients and cultured on nutrient agar, blood and MacConkey agar, at 37°C and incubated for 24 – 48 hours. The pH was adjusted to 7.4 and all media were sterilized by autoclaving at 121°C for 20 minutes. VITEK2 Compact identification kits was used to confirm the identification of the isolates and for antibiotic susceptibility test Results:  S. aureus were isolated in (43.48%) of isolated organisms and 31.88 % of the cases (n=22) were distributed between other staphylococcus species. S. pneumoniae in 10 cases (14.49%) and 10.15% of the cases (n=7) were distributed between other streptococcus species. All S. aureus isolates were resistant to penicillin but all S. aureus isolates were sensitive to quinupristin/dalfopristin, vancomycin, tigecycline, nitrofurantoin, rifampicin, and trimethoprim/sulfamethoxazole. S. pneumonia showed high rate of resistance to benzylpenicillin (100%) and oxacillin (100%) but the association trimethoprim/sulphamethoxazole showed moderate rate of resistance (50%) to S. pneumonia Conclusion: The prevalence of antibiotic resistance to gram positive bacteria continues to increase and is associated with significant mortality. The most prevalent organisms within gram positive bacteria were staphylococcus aureus followed by S. pneumoniae.