The status of the testicular lamina propria (LP) is associated with spermatogenesis. The aim of this study was to determine whether Raman spectroscopy (RS) could detect material components within the LP and predict spermatogenesis. Twenty adult male mice were divided into a busulfan-treated group (n=16 mice receiving a single injection of 50mgkg-1, i.p., busulfan) and a control group (n=4 mice receiving an equivalent volume of 0.9% saline solution injected i.p.). Mice were killed 2, 4, 6 and 8 weeks after injection of busulfan or saline solution (n=1 control and 4 busulfan-treated mice at each time point). The testicular tubules were assessed by RS and compared with histopathological observations. Control tubules had raw spectral intensities below 2000 arbitrary units, whereas busulfan tubules had strengthened intensities that peaked at Week 4 (absent spermatogenesis) and returned to normal levels at Week 8 (restored spermatogenesis). The change in the LP revealed by RS occurred before the change in spermatogenesis detected by histopathology. Correspondingly, the sensitivity/specificity of RS for distinguishing busulfan-treated and control tubules at 2, 4, 6 and 8 weeks were 65.00%/70.00%, 95.00%/100.00%, 40.00%/100.00% and 25.00%/95.00% respectively. Collectively, RS could be used to evaluate the status of the LP and as a complement to histopathological evaluation to predict tubules with the potential to develop spermatogenesis for infertile patients.