A high-performance liquid chromatography-ultraviolet detector (HPLC-UV) method has been used to quantify teicoplanin concentrations in human plasma. However, the limited analytical accuracy of previously bioanalytical methods for teicoplanin has given rise to uncertainty due to the use of an external standard. In this study, an internal standard (IS), polymyxin B, was applied to devise a precise, accurate, and feasible HPLC-UV method. The deproteinized plasma sample containing teicoplanin and an IS of acetonitrile was chromatographed on a C18 column with an acidic mobile phase consisting of NaH2PO4 buffer and acetonitrile (78:22, v/v) by isocratic elution and detection at 220 nm. The linearity was in the range 7.8–500 mg/L calculated by the ratio of the teicoplanin signal to the IS signal. This analytical method, validated by FDA guidelines with ICH Q2 (R1), was successfully applied to analyze the plasma samples of patients in the intensive care unit for treating serious resistant bacterial infectious diseases, such as those by methicillin-resistant Staphylococcus aureus and Enterococcus faecalis. The methods suggested the potential for use in routine clinical practice for therapeutic drug monitoring of teicoplanin, providing both improved accuracy and a wide range of linearity from lower than steady-state trough concentrations (10 mg/L) to much higher concentrations.