In the current study, a semi-automated, 96-well format, solid-phase extraction (SPE), analytical column-switching method for alendronate determination in human urine is developed, validated and applied to a bioequivalence study. The current protocol was a substantial improvement of an existing classical method. A robotic liquid handling system was employed to simplify and reduce the time of sample preparation procedure. Automated SPE was carried out using a 96-well cartridge plate and a vacuum control system. Urine samples were determined by applying a column-switching protocol with fluorescence detection. Analysis time, due to the column-switching procedure, was about half of the conventional LC approach (11.5 min instead of 21 min). The method application required the determination of alendronate in urine samples obtained from 96 healthy volunteers as part of a bioequivalence study of two 70 mg alendronate sodium tablets. All major pharmacokinetic parameters of the bioequivalence study were estimated and reported.