Coagulase-negative Staphylococcus (CoNS) has emerged as a significant primary pathogen, frequently determined in hospitalized individuals with immune deficiencies, and regularly with indwelling or implantable clinical devices. Most infections are nosocomial, and conservative infections can lead to a variety of chronic diseases, such as urinary tract infections (S. saprophyticus), infections brought on by medical devices, endocarditis, mediastinitis, meningitis, and bloodstream infections. Although many new species have been revealed in recent years, there is still a defect in clinical details for most of that genus. Additionally, interspecies variations that must be considered make certain species more pathogenic than others. Blood cultures and other samples are commonly contaminated because CoNS has the ability to colonize human skin. Determining cases where CoNS are causal agents rather than contaminants is hence the key diagnostic challenge. However, neither phenotypic nor genetic approaches have been successful in offering a satisfactory answer to this issue. MALDI-TOF MS (99.3% of strains properly identified) was the most effective method for speedy and precise CoNS differentiation. Vitek2 paired with partial tuf gene sequencing might be an alternative that yields 100% accurate strain identification when both techniques are used together. All of the staphylococcal subspecies under investigation, include S.capitis subspecies capitis as well as subspecies urealyticus, S.cohnii subspecies urealyticus, S.saprophyticus subspecies saprophyticus, and Staphylococcus hominis subspecies novobiosepticus and subspecies. As a result, staphylococci can be routinely identified by MALDI-TOF MS without incurring significant consumable expenses or requiring time-consuming DNA sequence analysis. The revolutionary high-quality RIDOM is superior to phenotypic techniques and the NCBI database. Staphylococci are easily identified using sequencing databases, even seldom isolated species and phenotypic differences. Another challenge facing CoNS is their comprehensive antimicrobial resistance profile is relevant to clinical practice, notably in medical facilities. Consequently, genuine infections brought on by CoNS require the usage of second-line antimicrobial medications the majority of the time.